Macrolide synthesis process and solid-state forms

ABSTRACT

Described are methods for making macrolides, and, in particular, a method for making optionally substituted 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide and derivatives thereof, as well as uses of macrolides to make medicaments, methods of treatment using macrolides, and methods for making intermediates that, among other uses, may be used to make macrolides. Also described are solvated and non-solvated crystalline forms of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, as well as methods for making such crystalline forms, medicaments comprising (or derived from) such crystalline forms, methods for making medicaments comprising (or derived from) such crystalline forms, methods of treatment using such crystalline forms, and kits comprising such crystalline forms.

This application is a continuation of U.S. patent application Ser. No.12/804,847, filed Jul. 30, 2010, now U.S. Pat. No. 8,263,753 issued Sep.11, 2012, which is a divisional of U.S. patent application Ser. No.11/828,404, filed Jul. 26, 2007, now U.S. Pat. No. 8,227,429 issued Jul.24, 2012, which claims priority to U.S. Provisional Patent ApplicationSer. No. 60/834,067, filed Jul. 28, 2006, and European PatentApplication No. 06118159.0, filed Jul. 31, 2006, the entire disclosureof each of which is hereby incorporated herein by this reference.

TECHNICAL FIELD

Described are methods for making macrolides, and, in particular, amethod for making optionally substituted20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide and derivatives thereof,as well as methods of treatment using such macrolides, uses of suchmacrolides to make medicaments, and methods for making intermediatesthat, inter alia, may be used to make macrolides. Also described aresolvated and non-solvated crystalline forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, as well as methods formaking such crystalline forms, medicaments comprising (or derived from)such crystalline forms, methods for making medicaments comprising (orderived from) such crystalline forms, methods of treatment using suchcrystalline forms, and kits comprising such crystalline forms.

BACKGROUND

Macrolides have long been known to be effective for treating infectiousdiseases in humans, livestock, poultry, and other animals. Earlymacrolides included 16-membered macrolides such as, e.g., tylosin A:

See, e.g., U.S. Pat. No. 4,920,103 (col. 5, lines 12-38). See also, U.S.Pat. No. 4,820,695 (col. 7, lines 1-32) and EP 0103465B1 (page 5, line3). Over the years, various tylosin derivatives have been developed withthe goal of enhancing antibacterial activity and selectivity.

Tylosin derivatives include, e.g., compounds discussed in U.S. Pat. No.6,514,946 that correspond in structure to Formula (I):

Here:

-   -   R¹ and R³ are each methyl, and R² is hydrogen; R¹ and R³ are        each hydrogen, and R² is methyl; or R¹, R², and R³ are each        hydrogen; and    -   R⁴ and R⁶ are each methyl, and R⁵ is hydrogen; R⁴ and R⁶ are        each hydrogen, and R⁵ is methyl; or R⁴, R⁵, and R⁶ are each        hydrogen.        Such compounds include, e.g.,        20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, which has the        following structure:

These compounds, and particularly20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, are believed to havepharmacokinetic and pharmacodynamic attributes for safe and effectivetreatment of, e.g., pasteurellosis, bovine respiratory disease, andswine respiratory disease. A discussion relating to the use of thesecompounds to treat livestock and poultry diseases is included in U.S.Pat. No. 6,514,946. That discussion is incorporated by reference herein.Applicants are not aware of any stable crystalline form of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide being described.

Various approaches for making macrolides have been reported.

In EP 0103465B1, e.g., Debono et al. discuss various process steps formaking compounds within their recited genus. These processes include,e.g., the following reduction:

Here, R, R¹, R², R³, and R⁴ are defined as various substituents. R, inparticular, is defined as a nitrogen-containing ring system that has upto 3 unsaturated or saturated rings that are optionally substituted.Debono et al. report that the preferred reducing agent is acyanoborohydride, and that sodium cyanoborohydride is “the reducingagent of choice.” Debono et al. also state that the solvent for thisreaction will normally be an inert polar solvent, such as a C₁-C₄alkanol. See page 6, lines 7-14. In a later-filed patent in the samepatent family, Debono et al. further discuss reductive amination ofvarious aldehyde compounds (including tylosin) with an amine. Sodiumcyanoborohydride and sodium borohydride are cited as suitable reducingagents, and anhydrous methanol is cited as a suitable solvent. See U.S.Pat. No. 4,820,695, col. 7, lines 60-68.

In U.S. Pat. No. 6,664,240, Phan et al. also discuss a reductiveamination:

Here, R^(p) ₂, R⁴, R⁷ and R⁸ are defined as various substituents. R⁷ andR⁸, in particular, are each defined as being independent substituents,or, alternatively, as together forming a 3- to 7-member heterocyclicring. Phan et al. discuss conducting this reaction with a borohydridereagent in an alcohol or acetonitrile solvent. Sodium borohydride andsodium cyanoborohydride are listed as example borohydride reagents; andmethanol, ethanol, and isopropanol are listed as example alcoholsolvents. See, e.g., col. 15, line 64 to col. 16, line 42; and col. 22,lines 41-49.

In EP 0240264B1, Tao et al. also discuss a reductive amination:

Here, R¹, R², R³, and R⁴ are defined as various substituents. R³ and R⁴,in particular, are defined as each being independent substituents, or,alternatively, as together forming a heterocyclic ring system having upto 3 optionally substituted rings. Tao et al. report that this reductionmay be achieved using formic acid as the reducing agent. Tao et al.further report that the solvent will ordinarily be an inert polarorganic solvent. Amyl acetate and acetonitrile are cited as examples ofsuch a solvent. See page 4, line 57 to page 5, line 10. See also, U.S.Pat. No. 4,921,947, col. 3, line 62 to col. 4, line 16.

In EP 0103465B1, Debono et al. discuss the following hydrolysisreaction:

Here, R, R¹, R², and R⁴ are defined as various substituents. Debono etal. report that this “hydrolysis can be effected using a strong aqueousmineral acid as hydrochloric or sulfuric acid, or a strong organic acidsuch as p-toluenesulfonic acid.” See, page 7, lines 3-8. In alater-filed patent of the same patent family, Debono et al. furtherdiscuss mycarose hydrolysis of C-20-modified derivatives of tylosin,macrocin, and DOMM using “well known” procedures for acidic hydrolysis.See U.S. Pat. No. 4,820,695, Col. 8, lines 35-43.

SUMMARY OF THE INVENTION

Described are processes for making macrolides, and, in particular,optionally substituted 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolideand derivatives thereof. Such processes include processes for making themacrolides themselves, as well as processes for making compounds that,inter alia, may be used as intermediates for making various macrolides.

Briefly, this disclosure is directed, in part, to a process for making amacrolide and salts thereof. The macrolide corresponds in structure toFormula (I):

Here:

-   -   As to R¹, R², and R³:        -   R¹ and R³ are each methyl, and R² is hydrogen,        -   R¹ and R³ are each hydrogen, and R² is methyl, or        -   R¹, R², and R³ are each hydrogen.    -   As to R⁴, R⁵, and R⁶:        -   R⁴ and R⁶ are each methyl, and R⁵ is hydrogen,        -   R⁴ and R⁶ are each hydrogen, and R⁵ is methyl, or        -   R⁴, R⁵, and R⁶ are each hydrogen.

In some embodiments, the process comprises reacting tylosin (e.g.,tylosin A or a salt thereof), a piperidinyl compound of Formula (II),and formic acid in the presence of a non-polar solvent. In theseembodiments, the piperidinyl compound of Formula (II) corresponds instructure to:

In some embodiments, the process comprises reacting a20-piperidinyl-tylosin compound with an acid. In these embodiments, the20-piperidinyl-tylosin compound corresponds in structure to Formula(III):

In some embodiments, the process comprises reacting a23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound withan acid. In these embodiments, the23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compoundcorresponds in structure to Formula (IV):

In some embodiments, the process comprises activating a23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound with anactivating agent to form an activated compound. In these embodiments,the 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compoundcorresponds in structure to Formula (V):

The activated compound (also referred to as a“23-L-20-piperidinyl-5-O-mycaminosyl-tylonolide compound”) correspondsin structure to Formula (VI):

And L is a leaving group.

In other embodiments, the process comprises reacting an activatedcompound of Formula (VI) with a piperidinyl compound of Formula (VII).In these embodiments, the piperidinyl compound of Formula (VII)corresponds in structure to:

In some embodiments, the process comprises a combination of the aboveembodiments to make a macrolide of Formula (I) or salt thereof.

In some embodiments, the process comprises one or more of the aboveembodiments to make, e.g., an amorphous, crystalline, co-crystalline, orsolvate form of the macrolide of Formula (I) or a salt thereof.

This disclosure also is directed, in part, to a process for making20-piperidinyl-tylosin compound of Formula (III) or a salt thereof. Insuch embodiments, the process comprises reacting tylosin (e.g., tylosinA), the piperidinyl compound of Formula (II), and formic acid in thepresence of a non-polar solvent.

This disclosure is also directed, in part, to a process for making the23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound ofFormula (IV) or a salt thereof. In these embodiments, the processcomprises reacting the 20-piperidinyl-tylosin compound of Formula (III)with HBr.

This disclosure is also directed, in part, to a process for making a23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound ofFormula (V) or a salt thereof. In these embodiments, the processcomprises reacting the23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound ofFormula (IV) with an acid.

This disclosure is also directed, in part, to a process for making anactivated compound of Formula (VI) or a salt thereof. In theseembodiments, the process comprises activating a23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound ofFormula (V) with an activating agent.

Also described are methods of using compounds of Formula (I) (andpharmaceutically acceptable salts thereof) prepared in accordance withthis disclosure in methods for treating a disease, such aspasteurellosis, swine respiratory disease, or bovine respiratorydisease. More specifically, this disclosure includes, in part, a methodthat comprises preparing a compound of Formula (I) (or apharmaceutically acceptable salt thereof) in accordance with one or moreof the cited methods, and then administering a therapeutically effectiveamount of the compound or salt to an animal in need of the treatment.This disclosure is also directed, in part, to using a compound ofFormula (I) (or a pharmaceutically acceptable salt thereof) prepared inaccordance with this invention to prepare a medicament, particularly amedicament for use in the above treatments.

Also described are crystalline forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

This disclosure is directed, in part, to a first crystalline20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide form (identified in thisdisclosure as the “Form I polymorph”). The Form I polymorph generallymay be characterized as having, e.g., at least one (and typically morethan one) of the following characteristics:

-   -   a. an FT-Raman spectrum comprising an absorption band at one or        more frequencies selected from the group consisting of about        2935, about 1633, about 1596, about 1712, about 1683, and about        781 cm⁻¹;    -   b. a powder X-ray diffraction spectrum comprising at least one        peak selected from the group consisting of 5.0 (±0.2) and 5.6        (±0.2) degrees 2Θ;    -   c. an attenuated total reflection infrared spectrum comprising        an absorption band at one or more frequencies selected from the        group consisting of about 2932, about 1711, about 1682, about        1635, about 1599, about 1442, about 1404, about 1182, about        1079, about 1053, about 1008, about 985, about 842, and about        783 cm⁻¹;    -   d. a melting point of from about 192 to about 195° C.; or    -   e. a melting enthalpy of about 57 J/g.

This disclosure is also directed, in part, to a second crystalline20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide form (identified in thisdisclosure as the “Form II polymorph”). The Form II polymorph generallymay be characterized as having, e.g., at least one (and typically morethan one) of the following characteristics:

-   -   a. an FT-Raman spectrum comprising an absorption band at one or        more frequencies selected from the group consisting of about        2929, about 1625, about 1595, about 1685, and 783 cm⁻¹;    -   b. a powder X-ray diffraction spectrum comprising a peak at 6.5        (±0.2) degrees 2Θ;    -   c. an attenuated total reflection infrared spectrum comprising        an absorption band at one or more frequencies selected from the        group consisting of about 2935, about 1736, about 1668, about        1587, about 1451, about 1165, about 1080, about 1057, about        1042, about 1005, about 981, about 838, and about 755 cm⁻¹;    -   d. a melting point of from about 113 to about 119° C.; or    -   e. a melting enthalpy of about 15 J/g.

This disclosure is also directed, in part, to a third crystalline20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide form (identified in thisdisclosure as the “Form III polymorph”). The Form III polymorphgenerally may be characterized as having, e.g., at least one (andtypically more than one) of the following characteristics:

-   -   a. an FT-Raman spectrum comprising an absorption band at one or        more frequencies selected from the group consisting of about        2943, about 2917, about 1627, about 1590, about 1733, about        1669, about 1193, about 1094, and about 981 cm⁻¹;    -   b. a powder X-ray diffraction spectrum comprising at least one        peak selected from the group consisting of 5.6 (±0.2) and 6.1        (±0.2) degrees 2Θ;    -   c. an attenuated total reflection infrared spectrum comprising        an absorption band at one or more frequencies selected from the        group consisting of about 2931, about 1732, about 1667, about        1590, about 1453, about 1165, about 1081, about 1057, about        1046, about 1005, about 981, about 834, and about 756 cm⁻¹;    -   d. a melting point of from about 107 to about 134° C.; or    -   e. a melting enthalpy of about 38 J/g.

This disclosure is also directed, in part, to a fourth crystalline20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide form (identified in thisdisclosure as the “Form IV polymorph”). The Form IV polymorph generallymay be characterized as having, e.g., at least one (and typically both)of the following characteristics:

-   -   a. an attenuated total reflection infrared spectrum comprising        an absorption band at one or more frequencies selected from the        group consisting of about 3559, about 2933, about 1743, about        1668, about 1584, about 1448, about 1165, about 1075, about        1060, about 1045, about 1010, about 985, about 839, and about        757 cm⁻¹; or    -   b. a melting point of from about 149 to about 155° C.

This disclosure is also directed, in part, to solvated crystalline formsof 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

In some embodiments, the solvated crystalline form comprises an ethylacetate (or “EtOAc”), ethanol, or diethyl ketone solvated crystallineform of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide; as well as anyother crystalline solvate that is isomorphic to the ethyl acetate,ethanol, or diethyl ketone solvated crystalline form. These crystallinesolvates are collectively identified in this disclosure as “S1crystalline solvates.”

In some embodiments, the solvated crystalline form comprises atert-butyl methyl ether (or “tBME”) solvated crystalline form of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, as well as any othercrystalline solvate that is isomorphic to the tBME solvated crystallineform. These crystalline solvates are collectively identified in thisdisclosure as “S2 crystalline solvates.”

In some embodiments, the solvated crystalline form comprises atetrahydrofuran (or “THF”) solvated crystalline form of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, as well as any othercrystalline solvate that is isomorphic to the THF solvated crystallineform. These crystalline solvates are collectively identified in thisdisclosure as “S3 crystalline solvates.”

In some embodiments, the solvated crystalline form comprises a methylacetate or ethyl formate solvated crystalline form of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, as well as any othercrystalline solvate that is isomorphic to the methyl acetate or ethylformate solvated crystalline form. These crystalline solvates arecollectively identified in this disclosure as “S4 crystalline solvates.”

This disclosure is also directed, in part, to compositions comprising20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. In these embodiments, anamount (generally at least a detectible quantity) of the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in the compositionconsists of one of the above-discussed solvated or non-solvatedcrystalline forms of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

This disclosure is also directed, in part, to a method for treating adisease, such as pasteurellosis, swine respiratory disease, or bovinerespiratory disease. The method comprises combining a therapeuticallyeffective amount of an above-discussed crystal-containing compositionwith at least one excipient to form a pharmaceutical composition, andadministering the pharmaceutical composition to an animal in need ofsuch treatment. In some such embodiments, e.g., a therapeuticallyeffective amount of the crystal-containing composition is dissolved in aliquid excipient(s) to form a solution that may, in turn, be used forparenteral or oral administration. In other such embodiments, atherapeutically effective amount of the crystal-containing compositionis suspended in a liquid excipient(s) to form a suspension that may, inturn, be used for parenteral or oral administration.

This disclosure is also directed, in part, to a use of a therapeuticallyeffective amount of an above-discussed crystal-containing composition toprepare a medicament for treating a disease (e.g., pasteurellosis, swinerespiratory disease, or bovine respiratory disease) in an animal.

This disclosure is also directed, in part, to a pharmaceuticalcomposition prepared by a process comprising combining at least oneexcipient with a therapeutically effective amount of an above-discussedcrystal-containing composition. In some such embodiments, e.g., atherapeutically effective amount of the crystal-containing compositionis dissolved in a liquid excipient(s) to form a solution that may, inturn, be used for parenteral or oral administration. In other suchembodiments, e.g., a therapeutically effective amount of thecrystal-containing composition is suspended in a liquid excipient(s) toform a suspension that may, in turn, be used for parenteral or oraladministration.

This disclosure is also directed, in part, to a kit. The kit comprises:a therapeutically effective amount of an above-discussedcrystal-containing composition, and instructions for combining thecrystal-containing composition with at least one excipient. The kit mayfurther (or alternatively) comprise additional components, such as,e.g., one or more excipients, one or more additional pharmaceutical orbiological materials, and/or one or more diagnostic tools.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an illustrative powder X-ray diffraction (“PXRD”) spectrumfor the Form I polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 2 shows an illustrative Fourier-transform Raman (“FT-Raman”)spectrum for the Form I polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 3 shows illustrative thermogravimetry coupled to Fourier transforminfrared spectroscopy (“TG-FTIR”) results for the Form I polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 4 shows illustrative differential scanning calorimetry (“DSC”)results for the Form I polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 5 shows illustrative dynamic vapor sorption (“DVS”) results for theForm I polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 6 shows an illustrative attenuated total reflection infrared(“ATR-IR”) spectrum (or “absorption band profile”) for the Form Ipolymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 7 shows an illustrative IR spectrum for a nujol suspensioncontaining the Form I polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 8 shows an illustrative PXRD spectrum for the Form II polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 9 shows an illustrative FT-Raman spectrum for the Form II polymorphof 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 10 shows illustrative TG-FTIR results for the Form II polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 11 shows illustrative DSC results for the Form II polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. The continuous linecorresponds to the first scan, and the dashed line corresponds to thesecond scan.

FIG. 12 shows illustrative DVS results for the Form II polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 13 shows an illustrative ATR-IR spectrum for the Form II polymorphof 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 14 shows an illustrative IR spectrum for a nujol suspensioncontaining the Form II polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 15 shows an illustrative PXRD spectrum for the Form III polymorphof 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 16 shows an illustrative FT-Raman spectrum for the Form IIIpolymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 17 shows illustrative TG results for the Form III polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 18 shows illustrative DSC results for the Form III polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. The continuous linecorresponds to the first scan, and the dashed line corresponds to thesecond scan.

FIG. 19 shows illustrative DVS results for the Form III polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 20 shows an illustrative ATR-IR spectrum for the Form III polymorphof 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 21 shows an illustrative IR spectrum for a nujol suspensioncontaining the Form III polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 22 shows an illustrative PXRD spectrum for the Form IV polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 23 shows illustrative DSC results for the Form IV polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 24 shows an illustrative ATR-IR spectrum for the Form IV polymorphof 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 25 shows an illustrative IR spectrum for a nujol suspensioncontaining the Form IV polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 26 shows an illustrative PXRD spectrum for an ethyl acetate S1crystalline solvate sample of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 27 shows an illustrative FT-Raman spectrum for an ethyl acetate S1crystalline solvate sample of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 28 shows illustrative TG-FTIR results for an ethyl acetate S1crystalline solvate sample of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 29 shows illustrative TG-FTIR results for an ethanol S1 crystallinesolvate sample of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 30 shows illustrative TG-FTIR results for a diethyl ketone S1crystalline solvate sample of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 31 shows an illustrative PXRD spectrum for a tBME S2 crystallinesolvate sample of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 32 shows an illustrative FT-Raman spectrum for a tBME S2crystalline solvate sample of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 33 shows illustrative TG-FTIR results for a tBME S2 crystallinesolvate sample of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 34 shows an illustrative PXRD spectrum for a THF S3 crystallinesolvate sample of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 35 shows an illustrative FT-Raman spectrum for a THF S3 crystallinesolvate sample of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 36 shows illustrative TG-FTIR results for a THF S3 crystallinesolvate sample of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 37 shows an illustrative PXRD spectrum for a methyl acetate S4crystalline solvate sample of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 38 shows an illustrative FT-Raman spectrum for a methyl acetate S4crystalline solvate sample of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 39 shows illustrative TG-FTIR results for an methyl acetate S4crystalline solvate sample of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

FIG. 40 shows illustrative TG-FTIR results for an ethyl formate S4crystalline solvate sample of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS A. Macrolides that May bePrepared

Compounds that may be prepared by the process of this invention includecompounds corresponding in structure to Formula (I):

Here:

-   -   R¹ and R³ are each methyl, and R² is hydrogen; R¹ and R³ are        each hydrogen, and R² is methyl; or R¹, R², and R³ are each        hydrogen; and    -   R⁴ and R⁶ are each methyl, and R⁵ is hydrogen; R⁴ and R⁶ are        each hydrogen, and R⁵ is methyl; or R⁴, R⁵, and R⁶ are each        hydrogen.

In some embodiments, the piperidinyl substituents of Formula (I) areidentical, i.e.:

IS THE SAME AS

In some such embodiments, e.g., both piperidinyl substituents arepiperidine (i.e., R¹, R², R³, R⁴, R⁵, and R⁶ are each hydrogen), suchthat the compound is 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide:

Such compounds include, for example:

Other compounds having identical piperidinyl substituents include:

In some embodiments, the piperidinyl substituents of Formula (I) are notidentical, i.e.:

IS DIFFERENT THAN

Compounds having different piperidinyl substituents include:

B. Macrolide Synthesis

This invention may be used to synthesize macrolides from materialsgenerally available in the art.

B-1. Preparation of 20-Piperidinyl-Tylosin Compound

In some embodiments, the macrolide synthesis begins by or includespreparing a 20-piperidinyl-tylosin compound, and particularly a compoundcorresponding in structure to Formula (III):

In some embodiments, R¹ and R³ are each methyl, and R² is hydrogen; orR¹ and R³ are each hydrogen, and R² is methyl. In other embodiments, R²,and R³ are each hydrogen, such that the compound corresponds instructure to:

The 20-piperidinyl-tylosin compound may be prepared from tylosin A and apiperidinyl compound via a reductive amination reaction using a reducingagent comprising formic acid (or “HCOOH”):

Where R¹, R², and R³ are each hydrogen, this reaction is as follows:

Tylosin A, piperidinyl compounds, and formic acid are commerciallyavailable.

The tylosin A reagent may, e.g., be pure (or at least essentially pure)tylosin A. Alternatively, as noted below in Section B-7, the tylosin Areagent may be part of a mixture, such as, e.g., a mixture comprisingtylosin A as well as one or more tylosin A derivatives, such as tylosinB, tylosin C, and/or tylosin D.

The tylosin A may be in the form of its free base, or, alternatively, inthe form of a salt. The tylosin A derivatives likewise are optionally inthe form of one or more salts. It is contemplated that a variety ofsalts may be suitable. In some embodiments, e.g., the salt comprises aphosphate salt. In other embodiments, the salt comprises a tartratesalt. In still other embodiments, the salt comprises a citrate orsulfate salt. Further discussion relating to salts may be found below inSection C.

The solvent may comprise one or more solvents. Although the solvent maycomprise one or more polar solvents in some embodiments, the solventpreferably instead comprises one or more non-polar solvents. A“non-polar solvent” is a solvent that does not ionize sufficiently to beelectrically conductive, and cannot (or at least essentially cannot)dissolve polar compounds (e.g., various inorganic salts), but candissolve nonpolar compounds (e.g., hydrocarbons and resins). In general,the solvent preferably is non-reactive with the reagents, products, andany other ingredients in the reaction mixture. The solvent may comprise,e.g., chloroform (or “CHCl₃”); tetrahydrofuran (or “THF”);dichloromethane (or “CH₂Cl₂” or “DCM” or “methylene chloride”); carbontetrachloride (or “CCl₄”); ethyl acetate (or “CH₃COOC₂H₅”); diethylether (or “CH₃CH₂OCH₂CH₃”); cyclohexane (or “C₆H₁₂”); or aromatichydrocarbon solvents, such as benzene (or “C₆H₆”), toluene (or“C₆H₅CH₃”), xylene (or “C₆H₄(CH₃)₂” or “dimethylbenzene” (including1,3-dimethylbenzene (or “m-xylene”), 1,2-dimethylbenzene (or“o-xylene”), or 1,4-dimethylbenzene (or “p-xylene”)), ethylbenzene, ormixtures thereof (e.g., mixtures of m-xylene, o-xylene, p-xylene, and/orethylbenzene). In some embodiments, the solvent comprisesdichloromethane, chloroform, or ethyl acetate. In other embodiments, thesolvent comprises xylene. In still other embodiments, the solventcomprises toluene. In some such embodiments, toluene is particularlypreferred because of its ease of use at typical reaction temperatures.

In some embodiments, the solvent comprises a mixture of solvents. Insome such embodiments, e.g., the solvent comprises a mixture of tolueneand DCM. Here, the toluene/DCM ratio may be, e.g., from about 1:1 toabout 100:1, or from about 5:1 to about 8:1 (v/v). In some of theseembodiments, the ratio is, e.g., about 8:1 (v/v). In others, the ratiois, e.g., about 5.3:1 (v/v).

To perform the amination, the tylosin A reagent, piperidinyl compound,formic acid (or a source of formic acid), and solvent normally arecharged to a reactor and mixed. These ingredients may generally becharged to the reactor in any sequence.

The reactor may comprise various reactor types. In some embodiments,e.g., the reactor is a stirred-tank reactor. Glass and glass-linedreactors are often preferred, although any composition stable whenexposed to the reaction mixture may be used. For example, stainlesssteel reactors generally may be used as well.

Typically, equimolar amounts of the tylosin A reagent, piperidinylcompound, and formic acid may be used. Normally, however, excess amountsof the piperidinyl compound and formic acid are used, relative to themolar amount of tylosin A reagent.

In some embodiments, from 1 to about 3 equivalents (or from 1.05 toabout 3 equivalents) of the piperidinyl compound are charged to thereactor. In some such embodiments, e.g., from 1.05 to about 1.2equivalents of piperidinyl compound are charged to the reactor. In othersuch embodiments, from about 1.07 to about 1.5 equivalents ofpiperidinyl compound are charged to the reactor. Here, e.g., about 1.3equivalents of the piperidinyl compound may be charged to the reactor.In some embodiments, the piperidinyl compound is charged to the reactorin two or more separate charges over time, preferably with thesubsequent charge(s) being less than the first charge. In someembodiments, e.g., the piperidinyl compound is charged to the reactor intwo charges, with the amount of the second charge being about 10% of thefirst charge. Applicants have discovered that this can be beneficial forincreasing conversion.

In some embodiments, from 1 to about 10 equivalents (or from 1.05 toabout 10 equivalents, from about 2 to about 5 equivalents, or from about2.5 to about 4.5 equivalents) of formic acid are used. In some suchembodiments, e.g., about 4.5 equivalents of formic acid are used. Inother such embodiments, from about 2.5 to about 4 equivalents of formicacid are used. For example, in some such embodiments, about 3.0equivalents of formic acid are used.

Typically, the amount of solvent is sufficient to, e.g., prevent (oressentially prevent) the reagents, products, and other ingredients inthe reaction mixture from sticking to the reactor, and promotehomogenous distribution of the reagents. In some embodiments, the amountof solvent is at least about 1 L per kg tylosin A reagent (or, where thetylosin A reagent is part of a mixture of tylosin A reagent andderivates thereof, per kg of the total tylosin mixture). The amount ofsolvent generally is less than about 40 L per kg tylosin A reagent (ortylosin mixture). In some embodiments, the amount of solvent is fromabout 2 to about 15 L (or from about 5 to about 15 L, from about 5 toabout 12 L, from about 5 to about 10 L, or from about 8 to about 10 L)per kg tylosin A reagent (or tylosin mixture). To illustrate, in somesuch embodiments, the solvent comprises toluene or a mixture of tolueneand DCM, and the amount of solvent is from about 8 to about 10 L per kgtylosin A reagent (or tylosin mixture). Here, e.g., the amount ofsolvent may be about 8 L per kg tylosin A reagent (or tylosin mixture).

At least a portion of the reaction (or the entire reaction) is typicallyconducted at greater than about 20° C., greater than about 25° C., orgreater than about 60° C. In general, at least a portion of the reaction(or the entire reaction) is conducted at a temperature that is notgreater than the boiling point of the solvent, and, more typically, isless than the boiling point. When, e.g., the solvent is toluene, atleast a portion of the reaction (or the entire reaction) is typicallyconducted at less than about 110° C. Illustrating further, when thesolvent is xylene, at least a portion of the reaction (or the entirereaction) is normally conducted at less than about 165° C. In general,at least a portion of the reaction (or the entire reaction) is conductedat from about 60 to about 95° C., from about 70 to about 85° C., fromabout 70 to about 80° C., or from about 75 to about 80° C. In someembodiments, e.g., the reaction temperature for at least a portion ofthe reaction (or the entire reaction) is about 80° C. In otherembodiments, e.g., the reaction temperature for at least a portion ofthe reaction (or the entire reaction) is about 76° C. Although lessertemperatures than the above ranges may be used, such temperatures tendto coincide with slower reaction rates. And, although greatertemperatures than the above ranges may be used, such temperatures tendto coincide with greater production of undesirable byproducts.

This reaction may be conducted over a wide range of pressures, includingatmospheric pressure, less than atmospheric pressure, and greater thanatmospheric pressure. It is typically preferred, however, to conduct thereaction at about atmospheric pressure. In preferred embodiments, thisreaction is conducted under an inert atmosphere (e.g., N₂).

The reaction time may depend on various factors including, e.g., thereaction temperature, characteristics of the solvent, relative amountsof the ingredients, and the desired conversion. In a batch reactor, thereaction time is generally at least about 1 minute, typically at leastabout 5 minutes, and more typically at least about 1 hour. The reactiontime is generally less than about 24 hours. In some embodiments, e.g.,the reaction time is from about 0.5 to about 12 hours, or from about 1to about 4 hours. In some such embodiments, the reaction time is about3.5 hours. In other such embodiments, the reaction time is from about 1to about 3 hours. In these embodiments, the reaction time may be, e.g.,about 2 hours. Although lesser reaction times than these ranges may beused, such reaction times tend to coincide with lesser conversions. And,although greater reaction times may be used, such reaction times tend tocoincide with greater production of impurities and inefficient use ofequipment and manpower.

Purification or isolation of the product may be achieved using, e.g.,various methods known in the art. Alternatively, the product may be usedin the next step without further purification or isolation.

B-2. Preparation of23-O-Mycinosyl-20-Piperidinyl-5-O-Mycaminosyl-Tylonolide Compound(Hydrolysis of Mycarosyloxy Substituent)

In some embodiments, the macrolide synthesis begins by or includespreparing a 23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound, and particularly a compound corresponding in structure toFormula (IV):

In some embodiments, R¹ and R³ are each methyl, and R² is hydrogen; orR¹ and R³ are each hydrogen, and R² is methyl. In other embodiments, R²,and R³ are each hydrogen such that the compound corresponds in structureto:

The 23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compoundmay be prepared via acid hydrolysis of a 20-piperidinyl-tylosincompound:

23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound

When R¹, R², and R³ are each hydrogen, this reaction is as follows:

The 20-piperidinyl-tylosin compound used in the above reaction may beprepared using a process discussed above in Section B-1, prepared usinga different process (e.g., a process using a borohydride as reducingagent), or obtained from a commercial vendor. In some embodiments, the20-piperidinyl-tylosin compound is prepared using a process discussedabove in Section B-1.

The acid may, e.g., be a strong mineral acid, such as hydrochloric acid(or “HCl”), nitric acid (or “HNO₃”), fluoroboric acid (or “HBF₄”),sulfuric acid (or “H₂SO₄”), phosphoric acid (or “H₃PO₄”), polyphosphonicacid (or “PPA”), or hydrobromic acid (or “HBr”); or a strong organicacid, such as p-toluenesulfonic acid or trifluoroacetic acid(“CF₃COOH”). In some embodiments, the acid comprises HCl. In otherembodiments, the acid comprises HBr. Use of HBr tends to coincide withless impurities in the product mixture relative to the product obtainedusing, e.g., HCl. In some embodiments, a mixture of acids (particularlya strong acid with another acid) is used.

In general, sufficient acid is mixed with the 20-piperidinyl-tylosincompound to hydrolyze (i.e., cleave) the mycarosyloxy substituent toform a hydroxyl group. Typically, the amount of acid will be at leastabout one equivalent, based on the amount of 20-piperidinyl-tylosincompound. In general, the acid is added to the reaction mixture in theform of a concentrated composition. The concentration typically is notgreater than about 50% (mass/vol), not greater than about 48%(mass/vol), from about 1 to about 30% (mass/vol), or from about 1 toabout 24% (mass/vol). In some embodiments, e.g., the acid is HBr, andthe concentration of the acid solution added to the reaction mixture isabout 24% (mass/vol). In some embodiments, the concentrated acidcomprises a mixture of acids, such as, e.g., HBr with another acid.

The ingredients may generally be charged to the reactor in any sequence.The reactor may comprise various reactor types. In some embodiments,e.g., the reactor is a stirred-tank reactor. Glass and glass-linedreactors are often preferred, although any composition stable whenexposed to the acidic reaction mixture may be used.

At least a portion of the hydrolysis (or the entire hydrolysis) normallyis conducted at a temperature that is greater than the freezing point ofthe mixture, allows the mixture to be stirred, and allows for themixture to be homogenous. A temperature of at least about 10° C. (orgreater than about 15° C., or greater than about 25° C.) is typicallypreferred. In general, the reaction temperature is not greater than theboiling point of the solvent (e.g., water), and typically is less thanthe boiling point. In some embodiments, at least a portion of thereaction (or the entire reaction) is conducted at a temperature that isnot greater than about 100° C. (or not greater than about 65° C.). Insome embodiments where the acid is HCl or HBr, the reaction temperatureover at least a portion of the reaction (or the entire reaction) is fromabout 20 to about 60° C. In some such embodiments, the reactiontemperature over at least a portion of the reaction (or the entirereaction) is not greater than about 40° C. In such instances, thereaction temperature over at least a portion of the reaction (or theentire reaction) may be, e.g., from about 20 to about 40° C., from about25 to about 40° C., or from about 30 to about 40° C. In otherembodiments where the acid is HCl or HBr, the reaction temperature overat least a portion of the reaction (or the entire reaction) is fromabout 45 to about 60° C., or from about 50 to about 56° C. Toillustrate, in such embodiments, the reaction temperature over at leasta portion of the reaction (or the entire reaction) may be, e.g., about56° C. Although greater temperatures than these ranges may be used, suchtemperatures tend to coincide with greater production of undesirablebyproducts. And, although lesser temperatures than these ranges may beused, such temperatures tend to coincide with slower reaction rates.Such rates, however, may still be suitable, given the ease with whichthis hydrolysis occurs.

The reaction mixture may be maintained at a temperature that is slightlyless than the desired reaction temperature while the acid is beingcharged to the reactor. In such embodiments, the temperature tends toincrease once the acid has been charged to the reactor.

This reaction may be conducted over a wide range of pressures, includingatmospheric pressure, less than atmospheric pressure, and greater thanatmospheric pressure. It is typically preferred, however, to conduct thereaction at about atmospheric pressure.

The reaction time will depend on various factors including, e.g., thereaction temperature, relative amounts of the ingredients, and thedesired conversion. In a batch reactor, the reaction time may be lessthan a minute, essentially spontaneous, or spontaneous. Generally,however, the reaction time is at least about 1 minute, more typically atleast about 5 minutes, and still more typically at least about 15minutes. Normally, the reaction time is less than about 3 hours. In someembodiments, e.g., the reaction time is from about 0.25 to about 2hours, from about 0.25 to about 1.5 hours, or from about 0.25 to about1.1 hours. Although lesser reaction times may be used, such reactiontimes may coincide with lesser conversions. And, although greaterreaction times may be used, such reaction times tend to coincide withgreater production of impurities and inefficient use of equipment andmanpower.

Purification or isolation of the product may be achieved using, e.g.,various methods known in the art. Alternatively, the product may be usedin the next step without further purification or isolation.

B-3. Preparation of23-Hydroxyl-20-Piperidinyl-5-O-Mycaminosyl-Tylonolide Compound(Hydrolysis of Mycinosyloxy Substituent)

In some embodiments, the macrolide synthesis begins by or includespreparing a 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound, and particularly a compound corresponding in structure forFormula (V):

In some embodiments, R¹ and R³ are each methyl, and R² is hydrogen; orR¹ and R³ are each hydrogen, and R² is methyl. In other embodiments, R¹,R², and R³ are each hydrogen:

The 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound maybe prepared via an acid hydrolysis reaction from a23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound:

Where R¹, R², and R³ are each hydrogen, this reaction is as follows:

The 23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compoundused in the above reaction may be prepared using a process discussedabove in Section B-2, prepared using a different process, or obtainedfrom a commercial vendor. In some preferred embodiments, the23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound isprepared using a process discussed above in Section B-2.

The acid may, e.g., be a strong mineral acid, such as hydrochloric acid,nitric acid, fluoroboric acid, sulfuric acid, phosphoric acid,polyphosphonic acid, or hydrobromic acid; or a strong organic acid, suchas p-toluenesulfonic acid or trifluoroacetic acid. In some embodiments,the acid comprises HCl. In some preferred embodiments, the acidcomprises HBr. As with the hydrolysis discussed above in Section B-2,this preference stems from the tendency of HBr to coincide with lessimpurities in the product mixture relative to, e.g., HCl. In someembodiments, a mixture of acids (particularly a mixture of a strong acidwith another acid) is used.

In embodiments where the mycinosyloxy hydrolysis occurs after the acidhydrolysis of mycarosyloxy discussed above in Section B-2, the acidsused in the mycinosyloxy and mycarosyloxy hydrolysis reactions may bedifferent, although it is generally more preferred for the acids to bethe same. In some embodiments, e.g., HCl is used in both reactions. Inother embodiments, HBr is used in both reactions.

In general, sufficient acid is mixed with the23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound tohydrolyze the mycinosyloxy substituent to form a hydroxyl group.Typically, the amount of acid will be greater than about one equivalent,based on the molar amount of23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound. Ingeneral, the acid is added to the reaction mixture in the form of aconcentrated composition. The concentration typically is not greaterthan about 50% (mass/vol), not greater than about 48% (mass/vol), fromabout 1 to about 30% (mass/vol), or from about 1 to about 24%(mass/vol). In some embodiments, e.g., the acid is HBr, andconcentration of the acid solution added to the reaction mixture isabout 24% (mass/vol). In some embodiments, the concentrated acidcomprises a mixture of acids, such as, e.g., HBr with another acid.

The ingredients may generally be charged to the reactor in any sequence.The reactor may comprise various reactor types. In some embodiments,e.g., the reactor is a stirred-tank reactor. Glass and glass-linedreactors are often preferred, although any composition stable whenexposed to the acidic reaction mixture may be used.

The mixture normally is maintained at a temperature that is greater thanthe freezing point of the mixture, allows the mixture to be stirred, andallows for the mixture to be homogenous. The reaction temperature overat least a portion of the reaction (or the entire reaction) preferablyis not greater than the boiling point of the solvent (e.g., water), andtypically is less than the boiling point. In general, at least a portionof the reaction (or the entire reaction) is conducted at a temperatureof at least about 10° C., greater than about 25° C., or at least about48° C. The reaction temperature over at least a portion of the reaction(or the entire reaction) typically is not greater than about 100° C., ornot greater than about 65° C. In some embodiments, e.g., the reactiontemperature over at least a portion of the reaction (or the entirereaction) is from about 10 to about 100° C. In some embodiments when theacid is HCl or HBr, the reaction temperature over at least a portion ofthe reaction (or the entire reaction) preferably is from about 48 toabout 60° C. In some such embodiments, e.g., the temperature over atleast a portion of the reaction (or the entire reaction) is from about55 to about 60° C. In other such embodiments, the temperature over atleast a portion of the reaction (or the entire reaction) is from about51 to about 57° C. (e.g., about 54° C.). In still other suchembodiments, the temperature over at least a portion of the reaction (orthe entire reaction) is from about 50 to about 56° C. Although lessertemperatures than these ranges may be used, such temperatures tend tocoincide with slower reaction rates. And, although greater temperaturesthan these ranges may be used, such temperatures tend to coincide withgreater production of undesirable byproducts.

As with the mycarosyloxy hydrolysis discussed above in Section B-2, themycinosyloxy hydrolysis reaction mixture may be maintained at atemperature that is slightly less than the desired reaction temperaturewhile at least a portion of the acid (or all the acid) is being chargedto the reactor.

This reaction may be conducted over a wide range of pressures, includingatmospheric pressure, less than atmospheric pressure, and greater thanatmospheric pressure. It is typically preferred, however, to conduct thereaction at about atmospheric pressure.

The reaction time depends on various factors including, e.g., thereaction temperature, relative amounts of the ingredients, and thedesired conversion. In a batch reactor, the reaction time is generallyat least about 1 minute, and more typically at least about 15 minutes.Typically, the reaction time is less than about 7 hours. In someembodiments, e.g., the reaction time is from about 0.5 to about 7 hours.In some such embodiments, e.g., the reaction time is from about 1 toabout 5 hours, or from about 3 to about 5 hours. Although lesserreaction times than these ranges may be used, such reaction times tendto coincide with lesser conversions. And, although greater reactiontimes may be used, such reaction times tend to coincide with greaterproduction of impurities and inefficient use of equipment and manpower.

When the mycinosyloxy hydrolysis occurs after the mycarosyloxyhydrolysis discussed above in Section B-2, the two reactions (i.e.,those described above in Section B-2 and this Section B-3) may becarried out as two discrete steps or as a single reaction. When thereactions are carried out as a single reaction, the reaction mixture maybe maintained at the same temperature or changed (typically increased)over time. If the reaction mixture is maintained at the sametemperature, the mixture normally is maintained at a temperature of atleast about 10° C., typically greater than about 25° C., more typicallyat least about 30° C., and still more typically at least about 45° C. Insome embodiments, the temperature is maintained at from about 10 toabout 100° C. In some such embodiments, e.g., the temperature is fromabout 48 to about 70° C. In other such embodiments, e.g., thetemperature is from about 50 to about 56° C. In other such embodiments,e.g., the temperature is from about 55 to about 60° C. In still othersuch embodiments, the temperature is from about 65 to about 70° C. Ifthe temperature of the mixture is increased over time, the temperatureof the mixture at the beginning of the hydrolysis normally is at leastabout 15° C., or at least about 25° C. As the reaction progresses fromthe mycarosyloxy hydrolysis to the mycinosyloxy hydrolysis, thetemperature preferably is increased to at least about 30° C., at leastabout 45° C., or from about 48 to about 70° C. In some such embodiments,the increased temperature is from about 50 to about 56° C. In other suchembodiments, the increased temperature is from about 55 to about 60° C.In still other such embodiments, the increased temperature is from about65 to about 70° C. In some embodiments, the reaction mixture ismaintained at a temperature that is slightly less than the desiredreaction temperature while the acid is being charged to the reactor. Inthose embodiments, the temperature tends to increase once the acid hasbeen charged to the reactor.

The total reaction time when the two hydrolysis reactions are combineddepends on various factors including, e.g., reaction temperature,relative amounts of the ingredients, and the desired conversion.Generally, however, the reaction time for the combined hydrolysisreactions in a batch reactor is at least about 4 hours. In someembodiments, the combined reaction time is from about 4 to about 6hours. In some such embodiments, e.g., the combined reaction time isabout 4 hours. Such a reaction time may be particularly suitable where,e.g., the reaction temperature is from about 65 to about 70° C. In otherembodiments, the reaction time is about 5 hours. Such a reaction timemay be particularly suitable where, e.g., the reaction temperature isfrom about 55 to about 60° C.

Purification or isolation of the product may be achieved using, e.g.,various methods known in the art. Alternatively, the product may be usedin the next step without further purification or isolation.

B-4. Preparing the Activated Compound

In some embodiments, the macrolide synthesis begins by or includespreparing an activated compound, and particularly a compound thatcorresponds in structure to Formula (VI):

In some embodiments, R¹ and R³ are each methyl, and R² is hydrogen; orR¹ and R³ are each hydrogen, and R² is methyl. In other embodiments, R¹,R², and R³ are each hydrogen, such that the compound corresponds instructure to:

L is a leaving group. In general, the leaving group is a group that maybe replaced with a piperidinyl group (normally via nucleophilicdisplacement) using piperidine in an amination reaction, such as theamination reaction discussed below in Section B-5. In some embodiments,e.g., L is iodo (—I), bromo (—Br), alkylsulfonate, and arylsulfonate.The alkylsulfonate and arylsulfonate are optionally substituted with oneor more substituents independently selected from the group consisting ofhalo, alkyl, and haloalkyl. In some such embodiments, e.g., L is iodo,bromo, methylsulfonate (or “—OS(O)₂CH₃” or “mesylate”),trifluoromethylsulfonate (or “—OS(O)₂CF₃” or “triflate”), or4-methylphenylsulfonate (or “p-toluenesulfonate” or “tosylate”). In someembodiments, L is iodo, and the activated compound corresponds instructure to:

or, when R¹, R², and R³ are each hydrogen, corresponds in structure to:

In some embodiments, L is mesylate, and the activated compoundcorresponds in structure to:

or, when R¹, R², and R³ each are hydrogen, corresponds in structure to:

In some embodiments, L is tosylate, and the activated compoundcorresponds in structure to:

or, when R¹, R², and R³ each are hydrogen, corresponds in structure to:

In some embodiments, L is triflate, and the activated compoundcorresponds in structure to:

or, when R¹, R², and R³ each are hydrogen, corresponds in structure to:

The activated compound may be prepared via an activation reaction from a23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound and anactivating agent (i.e., a compound comprising an electron withdrawinggroup):

Where R¹, R², and R³ are each hydrogen, this reaction is as follows:

The 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound usedin the above reaction may be prepared using a process discussed above inSection B-3, prepared using a different process, or obtained from acommercial vendor. In some preferred embodiments, the23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound isprepared using a process discussed above in Section B-3.

In some embodiments, L is iodo, and the activating agent is formed bymixing I₂ and triphenylphosphine:

Typically, this reaction is conducted in the presence of one or moresolvents. In general, the solvent is non-reactive with the reagents,products, and any other ingredients in the reaction mixture (although,as noted below, the solvent may, e.g., act as a helping base). Thesolvent may be, e.g., one or more of dichloromethane (“DCM”), acetone,acetonitrile (“ACN”), tert-butyl methyl ether (or “tBME”), toluene, andpyridine. In some embodiments, e.g., the solvent comprisestetrahydrofuran (“THF”). In other embodiments, the solvent comprisespyridine, which also may act as a helping base. In still otherembodiments, the solvent comprises dichloromethane. In some suchembodiments, e.g., the solvent comprises both dichloromethane andtoluene. In some embodiments, the ratio of dichloromethane to tolueneis, e.g., be at least about 1:1, from about 3:1 to about 10:1, or fromabout 3:1 to about 5:1. In some embodiments, at least a portion of thesolvent comprises solvent from a process step used during thepreparation and/or purification of the23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound.

In general, the amount of solvent is sufficient to, e.g., prevent (oressentially prevent) the reagents, products, and other ingredients inthe reaction mixture from sticking to the reactor, dissolve the reagents(particularly the 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound), and promote homogenous distribution of the reagents. Theamount of solvent typically is at least about 1 L per kilogram of the23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound. Theamount of solvent typically is not greater than about 100 L per kilogramof the 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound.In some embodiments, the amount of solvent is from about 5 to about 20 L(or from about 5 to about 15 L, or from about 10 to about 12 L) perkilogram of 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound. To illustrate, in some embodiments, the amount of solvent isabout 10 L DCM per kilogram. In other embodiments, the amount of solventis about 12 L DCM per kilogram. In still other embodiments, the solventis a mixture of DCM and toluene (about 4:1 vol/vol), and the amount ofsolvent is about 10 L per kilogram.

As indicated above, this reaction may be conducted in the presence of abase (or “helping base”). The base may be a single base, or acombination of bases. This base may comprise, e.g., triethylamine,pyridine, imidazole, potassium carbonate, and/or 4-dimethylaminopyridine(or “DMAP”). The presence of the base may increase the reaction rate. Insome embodiments, the base comprises pyridine. In some such embodiments,e.g., the activating agent comprises I₂ and triphenylphosphine. In otherembodiments, the base comprises imidazole. In other embodiments, thebase comprises a combination of potassium carbonate and4-dimethylaminopyridine. In some such embodiments, e.g., the activatingagent comprises p-toluenesulfonyl chloride. In some embodiments, thehelping base is attached to a solid support (e.g., a resin).

When a base is used, the molar amount of the base is typically at leastequivalent to the molar amount of23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound. In someembodiments, the amount of the base is at least 1.05 equivalents. Forexample, in some embodiments, the molar amount of the base is from about1.1 to about 10 equivalents, from about 1.1 to about 5 equivalents, orfrom about 1.1 to about 3 equivalents. In some such embodiments, themolar amount of base is from about 1.1 to about 1.4 equivalents (e.g.,about 1.15 or about 1.3 equivalents). In other such embodiments, themolar amount of the base is about 2.8 equivalents. When a combination ofbases is used, the total molar amount of base preferably falls withinthe ranges described above. For example, when the source of theactivating agent comprises p-toluenesulfonyl chloride, an example of acontemplated amount of base is about 1.5 equivalents of potassiumcarbonate and about 1.0 equivalents of 4-dimethylaminopyridine, based onthe amount of the 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound.

When the source of the activating agent is I₂ and triphenylphosphine,the I₂, triphenylphosphine, and base (if present) are typically firstcombined in the presence of solvent to form the activating agent beforethey are combined with the23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound. Thereactor in which the activating agent is formed may be the same reactorin which the activating agent is combined with the23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound.Alternatively, the activating agent may be formed in a differentreactor, and then charged to the reactor to which the23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound ischarged. The I₂ may be added in one or more doses to thetriphenylphosphine or vice versa. In some embodiments, the I₂ is addedto the triphenylphosphine in two or more portions (e.g., 5 portions) orvice versa. The portions may be equal or different amounts. Generally,the combination of the I₂ and triphenylphosphine takes place in thepresence of a solvent, which may, e.g., comprise the solvent(s) that isused in the substitution reaction. If a base (e.g., pyridine) ispresent, it is typically combined with the triphenylphosphine before theI₂ is added. The mixture preferably is maintained at from about 15 toabout 35° C. (or from about 20 to about 30° C., e.g., about 25° C.)during the addition of I₂ to the triphenylphosphine, and then maintainedat a temperature of from about 15 to about 35° C. (or from about 20 toabout 30° C., e.g., about 25° C.) after the addition for at least aboutone minute (e.g., about 2 minutes), or for at least about 5 minutes,from about 5 to about 60 minutes, or from about 30 to about 60 minutes(e.g., about 40 minutes). Afterward, the temperature preferably isadjusted to a temperature that is approximately equal to the temperatureat which the substitution reaction is to be initiated.

To perform the substitution reaction, equimolar amounts of the23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound andactivating agent generally may be used. Normally, however, an excess ofthe activating agent is used, and typically at least 1.05 equivalentsare used, based on the molar amount of23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound.

In some embodiments when the activating agent is formed from I₂ andtriphenylphosphine, the molar amounts of I₂ and triphenylphosphine areeach at least 1.05 equivalents, based on the molar amount of23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound. Forexample, in some such embodiments, the molar amounts of I₂ andtriphenylphosphine are each from 1.05 to about 10 equivalents, from 1.05to about 5 equivalents, or from 1.05 to about 3 equivalents. Althoughthe equivalents of each of I₂ and triphenylphosphine may be the same,the equivalents of triphenylphosphine typically exceeds the equivalentsof I₂. To illustrate, suitable molar amounts of I₂ andtriphenylphosphine may be about 2.5 and 2.6 equivalents, respectively,based on the molar amount of23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound. In otherembodiments, suitable molar amounts of I₂ and triphenylphosphine areabout 1.9 and 2.0 equivalents, respectively, based on the molar amountof 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound. Instill other embodiments, a suitable molar amount of I₂ is from 1.05 toabout 1.2 equivalents, based on the molar amount of23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound; and asuitable amount of triphenylphosphine is from about 1.09 to about 1.25equivalents, based on the molar amount of23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound. Forexample, suitable molar amounts of I₂ and triphenylphosphine may beabout 1.06 and about 1.13 equivalents, respectively, based on the molaramount of 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound. Exemplifying further, suitable molar amounts of I₂ andtriphenylphosphine may alternatively be about 1.2 and about 1.25equivalents, respectively, based on the molar amount of23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound.

In some embodiments when the activating agent is p-toluenesulfonylchloride, the molar amount of p-toluenesulfonyl chloride is from about1.1 to about 10 equivalents, from about 1.2 to about 5 equivalents, orfrom about 1.2 to about 3 equivalents, based on the molar amount of23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound. Asuitable molar amount of p-toluenesulfonyl chloride may be, e.g., 1.2equivalents.

The substitution reaction may be conducted over a wide range ofpressures, including atmospheric pressure, less than atmosphericpressure, and greater than atmospheric pressure. It is typicallypreferred, however, to conduct the reaction at about atmosphericpressure.

The reaction temperature for at least a portion of the substitutionreaction (or the entire substitution reaction) typically is greater thanthe freezing point of the solvent. In general, the reaction temperatureover at least a portion of the substitution (or the entire substitution)is not greater than the boiling point of the solvent, and typically isless than the boiling point. In some embodiments, e.g., the solvent isdichloromethane, and at least a portion of the reaction (or the entirereaction) is conducted at a temperature that is not greater than about45° C. In some embodiments, at least a portion of the reaction (or theentire reaction) is conducted at a temperature that is not greater thanabout 32° C., or not greater than about 25° C. In some such embodiments,e.g., at least a portion of the reaction (or the entire reaction) isconducted at from about −10° C. to about 25° C. For example, in someembodiments, at least a portion of the reaction (or the entire reaction)is conducted at from about zero to about 20° C., or from about 12 toabout 18° C. (e.g., about 13° C.). In other embodiments, at least aportion of the reaction (or the entire reaction) is conducted at fromabout −10° C. to about 45° C., or from about 25 to about 45° C. In stillother embodiments, at least a portion of the reaction (or the entirereaction) is conducted at from about −10 to about 0° C., or from about−6 to about −5° C. Although lesser temperatures than these ranges may beused, such temperatures tend to coincide with slower reaction rates.And, although greater temperatures than these ranges may be used, suchtemperatures tend to coincide with greater production of undesirablebyproducts. Use of some sources of the activating agent (e.g.,toluenesulfonyl chloride), however, may allow for greater temperaturesto be used (e.g., from about 25 to about 45° C.). When the activatingagent is iodine, it is typically preferred to conduct the substitutionreaction within a temperature range that does not produce anunacceptable level of impurities resulting from di-iodination of the23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound.

The reaction time depends on various factors including, e.g., thereaction temperature, characteristics of the solvent, relative amountsof the ingredients, and the desired conversion. In a batch reactor, thetotal reaction time is typically at least about 1 minute, and moretypically at least about 45 minutes. In general, the total reaction timeis less than about 24 hours. In some embodiments, e.g., the totalreaction time is less than about 5 hours. To illustrate, in someembodiments, the reaction time is from about 45 minutes to about 5hours, or from about 1 to about 3 hours. In some such embodiments, e.g.,the reaction time is from about 2 to about 3 hours, or from about 2 toabout 2.5 hours (e.g., about 2 or about 2.2 hours). In otherembodiments, the reaction time is from about 5 to about 10 hours, fromabout 6 to about 10 hours, from about 7 to about 10 hours, or from about7 to about 8 hours. Although lesser reaction times than these ranges maybe used, such reaction times tend to coincide with lesser conversions.And, although greater reaction times may be used, such reaction timestend to coincide with greater production of impurities and inefficientuse of equipment and manpower.

Due to the exothermic nature of the substitution reaction, in someembodiments (particularly those using a batch reactor), the23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound iscombined with the activating agent over time (or in multiple separatedoses) rather than all at once. In some embodiments, this occurs over aperiod of at least one minute, at least 5 minutes, from about 5 to about60 minutes, or from about 30 to about 60 minutes (e.g., about 50minutes). To illustrate, in some embodiments, the substitution reactionis conducted at a maximum temperature of about 25° C., and the dosing ofthe activating agent occurs over from about 0.5 to about 1 hour,followed by an additional reaction time of about 1 hour. In otherembodiments, the substitution reaction is conducted at a maximumtemperature of about −5° C., and the dosing of the activating agentoccurs over from about 0.7 to about 1 hour, followed by an additionalreaction time of about 7 hours.

In some embodiments, the substitution reaction is quenched to inactivateany residual iodine, and, therefore, reduce (and preferably prevent)by-product formation due to such residual iodine. For example, in somesuch embodiments, the reaction is quenched with aqueous sodium sulfite(i.e., Na₂SO₃). Purification or isolation of the product may be achievedusing, e.g., various methods known in the art. Alternatively, theproduct may be used in the next step without further purification orisolation.

Both the activating agent formation reaction and the substitutionreaction may be conducted in various reactor types. In some embodiments,e.g., the reactor is a stirred-tank reactor. The reactor may be made ofany composition that remains stable when exposed to the reactionmixture. Such materials include, e.g., various materials, such as glass(including glass-lining) or stainless steel.

B-5. Preparing the Macrolide

As noted above, the macrolides prepared in accordance with thisinvention correspond in structure to Formula (I):

Here:

-   -   As to R¹, R², and R³:        -   R¹ and R³ are each methyl, and R² is hydrogen;        -   R¹ and R³ are each hydrogen, and R² is methyl; or        -   R¹, R², and R³ are each hydrogen.    -   As to R⁴, R⁵, and R⁶:        -   R⁴ and R⁶ are each methyl, and R⁵ is hydrogen;        -   R⁴ and R⁶ are each hydrogen, and R⁵ is methyl; or        -   R⁴, R⁵, and R⁶ are each hydrogen.            In some embodiments, R¹, R², R³, R⁴, R⁵, and R⁶ are each            hydrogen:

In some such embodiments, e.g., the compound corresponds in structure tothe following formula:

In some embodiments, the preparation of the macrolide begins by orincludes an amination reaction of an activated compound with apiperidinyl compound:

Where R¹, R², R³, R⁴, R⁵, and R⁶ are each hydrogen, this reaction is asfollows:

The activated compound used in this reaction may be prepared using aprocess discussed above in Section B-4, or a different process, orobtained from a commercial vendor. In some embodiments, the activatedcompound is prepared using a process discussed herein in Section B-4.

Typically, this reaction is conducted in the presence of one or moresolvents. In general, the solvent is non-reactive with the reagents(e.g., the activated compound), products, and any other ingredients inthe reaction mixture. The solvent may be, e.g., acetonitrile (or“CH₃CN”); chloroform; dichloromethane; tetrahydrofuran; a ketonesolvent, such as acetone (or “CH₃COCH₃”); a hydrocarbon solvent, such asan aromatic hydrocarbon solvent (e.g., toluene or xylene); or a base,such as pyridine or piperidine. In some embodiments, the solventcomprises acetonitrile, tetrahydrofuran, and/or dichloromethane. In someembodiments, the solvent comprises xylene. In some embodiments, at leasta portion of the solvent comprises solvent from a process step usedduring the preparation and/or purification of the activated compound.

The amount of solvent can vary widely, from no solvent at all to amountsthat create diluted reaction mixtures. Typically, the amount of solventis sufficient to, e.g., prevent (or essentially prevent) the reagents,products, and other ingredients in the reaction mixture from sticking tothe reactor, and promote homogenous distribution of the reagents. Insome embodiments, sufficient solvent is present such that the combinedamount of activating reagent and macrolide is from about 5 to about 50%(mass/volume) of the reaction mixture. In some embodiments, the amountof solvent is at least about 1 L per kilogram activated compound. Insome such embodiments, e.g., the amount of solvent is from about 1 toabout 100 L (or from about 1 to about 20 L) per kilogram of activatedcompound. To illustrate, in some embodiments, about 5 L of solvent(e.g., xylene or tetrahydrofuran) are used per kilogram of activatedcompound. Illustrating further, in other embodiments, about 10 L ofsolvent (e.g., acetonitrile) are used per kilogram of activatedcompound.

In some embodiments, the amination is conducted in the presence of abase. In some embodiments, the base comprises an un-hydrated base. Thebase may be, e.g., potassium carbonate (or “K₂CO₃”), sodium carbonate(or “Na₂CO₃”), or a tertiary amine. The presence of such a base tends tocoincide with a greater reaction rate and less impurities. It isbelieved that such advantages may stem from the base deprotonatingprotonated piperidinyl compound. The base preferably is not so strong asto cause hydrolysis of the lactone in the macrolide core. Generally,equimolar amounts of the activated compound and base may be used.Normally, however, an excess of base is used. In some embodiments, atleast 1.05 (or from about 1.1 to about 50, from about 2 to about 30,from about 2 to about 20, or from about 2 to about 10) equivalents ofbase are used, based on the molar amount of activated compound chargedto the reactor. In some such embodiments, about 6.2 equivalents of baseare used. In other some such embodiments, about 10 equivalents of baseare used. In still other such embodiments, from about 1.1 to about 10(or from about 2 to about 8, or from about 4 to about 6) equivalents ofbase are used, based on the molar amount of activated compound chargedto the reactor. To illustrate, a suitable amount of base may be, e.g.,about 5 equivalents.

To perform the amination, the activated compound, piperidinyl compound,and solvent, as well as any base (to the extent present), are normallycharged to a reactor and mixed. These ingredients generally may becharged to the reactor in any sequence. The reactor may comprise variousreactor types. In some embodiments, e.g., the reactor is a stirred-tankreactor. Glass, glass-lined, and stainless steel reactors are oftenpreferred, although any composition stable when exposed to the reactionmixture may be used.

Generally, equimolar amounts of the activated compound and piperidinylcompound may be used. Normally, however, an excess of the piperidinylcompound is used. In some embodiments, at least 1.05 (or from about 1.1to about 50, from about 2 to about 30, from about 2 to about 20, or fromabout 2 to about 10) equivalents of the piperidinyl compound are used,based upon the molar amount of activated compound charged to thereactor. In some such embodiments, about 10 equivalents of thepiperidinyl compound are used. In other such embodiments, from about 2to about 8 (or from about 4 to about 6) equivalents are used, based uponthe molar amount of activated compound charged to the reactor. Toillustrate, a suitable amount of piperidinyl compound may be, e.g.,about 4.7 equivalents. A contemplated suitable amount of piperidinylcompound also may be, e.g., about 5.7-5.8 equivalents.

At least a portion of the reaction (or the entire reaction) normally isconducted at greater than about 20° C., or greater than about 25° C. Theoptimal reaction temperature depends on, e.g., the solvent. Typically,at least a portion of the reaction (or the entire reaction) is conductedat a temperature that is not greater than the boiling point of thesolvent, and typically is less than the boiling point. In general, atleast a portion of the reaction (or the entire reaction) is conducted atfrom about 50 to about 110° C. In some embodiments, e.g., at least aportion of the reaction (or the entire reaction) is conducted at fromabout 60 to about 110° C., or from about 75 to about 110° C. Toillustrate, when the solvent comprises acetonitrile or toluene, asuitable contemplated reaction temperature for at least a portion of thereaction (or the entire reaction) is from about 78° C. to about 110° C.(e.g., about 78° C.). To illustrate further, when the solvent comprisesxylene, a suitable contemplated reaction temperature for at least aportion of the reaction (or the entire reaction) is from about 95 toabout 105° C., and a suitable reaction time for the reaction is about 15hours. In other embodiments, the solvent comprises tetrahydrofuran, andat least a portion of the reaction (or the entire reaction) is conductedat from about 55 to about 75° C. Although lesser temperatures than theseranges may be used, such temperatures tend to coincide with slowerreaction rates. And, although greater temperatures than these ranges maybe used, such temperatures tend to coincide with greater production ofundesirable byproducts. Typically, lesser temperatures may be used withsolvents having greater polarities. The temperature can be adaptedaccordingly by one skilled in the art.

In some embodiments, the amination reaction is conducted at more thanone temperature. For example, the reaction may be conducted at onetemperature initially, and then slowly increased to another temperatureas the reaction progresses.

The amination may be conducted over a wide range of pressures, includingatmospheric pressure, less than atmospheric pressure, and greater thanatmospheric pressure. It is typically preferred, however, to conduct thereaction at about atmospheric pressure.

The reaction time depends on various factors including, e.g., thereaction temperature, characteristics of the solvent, relative amountsof the ingredients, and the desired conversion. In a batch reactor, thereaction time is generally at least about 1 minute, at least about 5minutes, or at least about 45 minutes. The reaction time is generally nogreater than about 24 hours. In some embodiments, the reaction time isfrom about 2 to about 15 hours. In other embodiments, the reaction timeis from about 1 to about 5 hours, from about 2 to about 4 hours, or fromabout 2 to about 3 hours (e.g., about 2.5 hours). In other suchembodiments, the reaction time is from about 6 to about 15 hours.Although lesser reaction times than these ranges may be used, suchreaction times tend to coincide with lesser conversions.

Further purification or isolation of the product may be achieved using,e.g., various methods known in the art.

B-6. Examples of Contemplated Reaction Schemes

This invention contemplates any processes that use any of the abovereactions. In some embodiments, the process will comprise one of theabove reactions. In other embodiments, the process will comprise two,three, four, or all the above reactions. The following Scheme Igenerically illustrates a scenario where all the above reactions areused:

Here:

-   -   As to R¹, R², and R³:        -   R¹ and R³ are each methyl, and R² is hydrogen;        -   R¹ and R³ are each hydrogen, and R² is methyl; or        -   R¹, R², and R³ are each hydrogen.    -   As to R⁴, R⁵, and R⁶:        -   R⁴ and R⁶ are each methyl, and R⁵ is hydrogen;        -   R⁴ and R⁶ are each hydrogen, and R⁵ is methyl; or        -   R⁴, R⁵, and R⁶ are each hydrogen.    -   L is a leaving group.

The following Scheme II generically illustrates the above scenario wherethe non-polar solvent in the reductive amination comprises toluene; theacids in the hydrolysis reactions comprise HBr; the source of theactivating agent comprises I₂, triphenylphosphine, and pyridine; and thefinal amination reaction mixture comprises potassium carbonate:

The following Scheme III generically illustrates the scenario of SchemeI where the two hydrolysis reactions are conducted without stopping thefirst hydrolysis or isolating product from the first hydrolysis beforeconducting the second hydrolysis:

The following Scheme IV generically illustrates the scenario of Scheme Iwhere the non-polar solvent in the reductive amination comprisestoluene; the acids in the hydrolysis reactions comprise HBr; the firsthydrolysis is not stopped and the product of the first hydrolysis is notisolated before conducting the second hydrolysis; the source of theactivating agent comprises I₂, triphenylphosphine, and pyridine; and thefinal amination reaction mixture comprises potassium carbonate:

The following Scheme V generically illustrates a 2-stage scenario ofScheme I where the non-polar solvent in the reductive aminationcomprises toluene; the acids in the hydrolysis reactions comprise HBr;the reductive amination and first hydrolysis are not stopped and theproducts of the reductive amination and first hydrolysis are notisolated before conducting the second hydrolysis; the source of theactivating agent comprises I₂, triphenylphosphine, and pyridine; thefinal amination reaction mixture comprises potassium carbonate; and theactivation reaction is not stopped and the product of the activationreaction is not isolated before conducting the final amination reaction:

In general, the tylosin reagent used in the processes of this inventioncomprises tylosin A (or a salt thereof):

Although this invention contemplates the use of pure (or at leastessentially pure) tylosin A (or a salt thereof), various commerciallyavailable tylosin compositions additionally or alternatively maycomprise one or more derivatives of tylosin A, including:

In general, these derivatives, if present, are present in only smallamounts. In some embodiments, the weight ratio of tylosin A to the totalcombined amount of tylosin A derivatives in the composition is at leastabout 1:1. In some such embodiments, e.g., the ratio is at least about4:1, at least about 10:1, at least about 95:5, at least about 98:2, orat least about 99:1. In other such embodiments, about 100% (by weight)of the tylosin compounds (i.e., tylosin A and tylosin A derivatives) inthe composition consists of tylosin A. Other embodiments arecontemplated wherein the tylosin A makes up less than 50% (by weight) ofthe tylosin compounds in the composition. To illustrate, in some suchembodiments, the weight ratio of tylosin D to the total combined amountsof tylosin A and other tylosin A derivatives is at least about 1:1, atleast about 4:1, at least about 9:1, at least about 95:5, at least about98:2, or at least about 99:1. In other such embodiments, about 100% (byweight) of the tylosin compounds in the composition consists of tylosinD.

The methods discussed above for making20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide from tylosin A generallycan be used (and, to the extent desirable, modified) for making20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide from tylosin B, C, and/orD in addition to (or instead of) tylosin A.

Tylosin B, e.g., has a hydroxyl instead of a mycarosyl substituent onthe 5-mycanimosyl. Thus, a 20-piperidinyl intermediate derived fromtylosin B does not require the first hydrolysis reaction discussed abovein Section B-2. To the extent such an intermediate is exposed to thehydrolysis method discussed in Section B-2, the intermediate willgenerally remain non-reactive or begin hydrolyzing at the23-mycinosyloxy substituent.

Tylosin C has a hydroxyl rather than a methoxy at the 3-position of the23-mycinosyloxy substituent. This difference generally has no effectwith respect to the above-described methods. The sugar normally willcleave (i.e., hydrolyze) in the same manner and under the sameconditions as the 23-mycinosyloxy of tylosin A during the hydrolysisdescribed above in Section B-3.

Tylosin D has a hydroxyl rather than a carbonyl at the 20-position. Thishydroxyl is generally not transformed into a piperidinyl using thereductive amination method described above in Section B-1. Depending onthe reaction conditions, however, it may become activated during theactivation reaction described above in Section B-4, and then aminatedwith piperidine along with the 23-position during the amination methoddescribed above in Section B-5.

C. Salts of Intermediates and Macrolides

This invention may be used to prepare macrolide compounds orintermediates both in the form of free compounds and in the form ofsalts. In addition, the reagents used in this invention may be in theform of salts. Salts may be, e.g., acid addition salts. In general, anacid addition salt can be prepared using any inorganic or organic acid.Depending on the particular compound (and/or its crystalline structure),a salt of a compound may be advantageous due to one or more of thesalt's chemical or physical properties, such as stability in differingtemperatures and humidities, or a desirable solubility in water, oil, orother solvent. In some instances, a salt of a compound also may be usedas an aid in the isolation or purification of a compound. In someembodiments (particularly where the salt is intended to be administeredto an animal, as opposed to, e.g., being used in an in vitro context),the salt is pharmaceutically acceptable.

Salts can typically be formed by, e.g., mixing the free macrolide orintermediate compound with an acid using various known methods in theart. Examples of often suitable inorganic acids include hydrochloric,hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoricacid. Examples of often suitable organic acids include, e.g., aliphatic,cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, andsulfonic classes of organic acids. Specific examples of often suitableorganic salts include cholate, sorbate, laurate, acetate,trifluoroacetate (or “CF₃COOH” or “TFA”), formate, propionate,succinate, glycolate, gluconate, digluconate, lactate, malate, tartrate(and derivatives thereof, such as dibenzoyltartrate), citrate,ascorbate, glucuronate, maleate, fumarate, pyruvate, aspartate,glutamate, benzoate, anthranilic acid, mesylate, stearate, salicylate,p-hydroxybenzoate, phenylacetate, mandelate (and derivatives thereof),embonate (pamoate), ethanesulfonate, benzenesulfonate, pantothenate,2-hydroxyethanesulfonate, sulfanilate, cyclohexylaminosulfonate,β-hydroxybutyric acid, galactarate, galacturonate, adipate, alginate,butyrate, camphorate, camphorsulfonate, cyclopentanepropionate,dodecylsulfate, glycoheptanoate, glycerophosphate, heptanoate,hexanoate, nicotinate, 2-naphthalesulfonate, oxalate, palmoate,pectinate, 3-phenylpropionate, picrate, pivalate, thiocyanate, tosylate,and undecanoate. In some embodiments, the salt comprises ahydrochlorate, trifluoroacetate, mesylate, tosylate, tartrate, orcitrate salt.

D. Crystalline Forms of 20,23-Dipiperidinyl-5-O-Mycaminosyl-Tylonolide

The chemical and physical properties of macrolides, and particularly20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, are often important intheir commercial development. These properties include, for example: (1)physical stability; (2) chemical stability; (3) packing properties, suchas molar volume, density, and hygroscopicity; (4) thermodynamicproperties, such as melting temperature, vapor pressure, and solubility;(5) kinetic properties, such as dissolution rate and stability(including stability at ambient conditions, especially to moisture andunder storage conditions); (6) surface properties, such as surface area,wettability, interfacial tension, and shape; (7) mechanical properties,such as hardness, tensile strength, compactibility, handling, flow, andblend; (8) filtration properties; and (9) chemical purity. Theseproperties can affect, e.g., processing and storage of pharmaceuticalcompositions comprising 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.While Applicants believe that all the solid-state forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide described in thisdisclosure are therapeutically effective, solid-state forms that providean improvement in one or more of the above-listed properties relative toother solid-state forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide are generally desirable,as are solid-state forms that may be used as intermediates in processesfor making the desired solid-state forms.

In accordance with the invention, several crystalline forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide have been prepared. Thesecrystalline forms generally possess one or more of the above-describedadvantageous chemical and/or physical properties relative to othersolid-state forms of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolideand/or are useful as intermediates in the preparation of one or moreother solid-state forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. Specific crystallineforms of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide that have beendiscovered include the following:

-   -   (1) A first anhydrous and non-solvated crystalline form of        20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide possessing unique        properties relative to other solid-state forms of        20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (identified in        this disclosure as the “Form I polymorph”);    -   (2) a second anhydrous and non-solvated crystalline form of        20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide possessing unique        properties relative to other solid-state forms of        20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (identified in        this disclosure as the “Form II polymorph”);    -   (3) a third anhydrous and non-solvated crystalline form of        20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide possessing unique        properties relative to other solid-state forms of        20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (identified in        this disclosure as the “Form III polymorph”);    -   (4) a fourth anhydrous and non-solvated crystalline form of        20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide possessing unique        properties relative to other solid-state forms of        20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (identified in        this disclosure as the “Form IV polymorph”);    -   (5) isomorphic ethyl acetate, ethanol, and diethyl ketone        solvated crystalline forms of        20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (collectively        identified in this disclosure as “S1 crystalline solvates”);    -   (6) a tert-butyl methyl ether solvated crystalline form of        20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (identified in        this disclosure as an “S2 crystalline solvate”);    -   (7) a tetrahydrofuran solvated crystalline form of        20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (identified in        this disclosure as an “S3 crystalline solvate”); and    -   (8) isomorphic methyl acetate and ethyl formate solvated        crystalline forms of        20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (collectively        identified in this disclosure as “S4 crystalline solvates”).

In some embodiments, the invention is directed to the Form I polymorphof 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. Illustrative methodsfor making the Form I polymorph include, e.g., those shown in Examples 3(Part F) and 12-16. Based on Applicants' observations, it is believedthat the Form I polymorph generally possesses greater stability atambient temperature than the other above-listed solid-state forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, particularly in theabsence of solvent. In many embodiments, it is desirable to use asolid-state form, such as the Form I polymorph, that typically does notrequire special processing or storage conditions, and avoids the needfor frequent inventory replacement. For example, selecting a solid-stateform that is physically stable during a manufacturing process (such asduring milling to obtain a material with reduced particle size andincreased surface area) can avoid the need for special processingconditions and the increased costs generally associated with suchspecial processing conditions. Similarly, selection of a solid-stateform that is physically stable over a wide range of storage conditions(especially considering the different possible storage conditions thatcan occur during the lifetime of a20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide product) can help avoidpolymorphic or other degradative changes in the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide that can lead to productloss or deterioration of product efficacy. Thus, the selection of asolid-state form having greater physical stability provides a meaningfulbenefit over less-stable solid-state forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. The Form I polymorphalso tends to exhibit less water uptake than other solid-state forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide under, e.g., ambientconditions (e.g., 25° C.). It is further hypothesized that the Form Ipolymorph exhibits advantageous packing properties, thermodynamicproperties, kinetic properties, surface properties, mechanicalproperties, filtration properties, or chemical purity relative to othersolid-state forms of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

The Form I polymorph may be identified using various analyticaltechniques. In some embodiments, the Form I polymorph is defined ashaving one (and typically two, three, four, or all five) of thefollowing characteristics:

-   -   a. an FT-Raman spectrum comprising an absorption band at one or        more frequencies selected from the group consisting of about        2935, about 1633, about 1596, about 1712, about 1683, and about        781 cm⁻¹;    -   b. a powder X-ray diffraction spectrum comprising at least one        peak selected from the group consisting of 5.0 (±0.2) and 5.6        (±0.2) degrees 2Θ;    -   c. an attenuated total reflection infrared spectrum comprising        an absorption band at one or more frequencies selected from the        group consisting of about 2932, about 1711, about 1682, about        1635, about 1599, about 1442, about 1404, about 1182, about        1079, about 1053, about 1008, about 985, about 842, and about        783 cm⁻¹.    -   d. a melting point of from about 192 to about 195° C.; or    -   e. a melting enthalpy of about 57 J/g.

In some embodiments, the Form I polymorph is defined as having anFT-Raman spectrum comprising an absorption band at about 2935 cm⁻¹. Inother embodiments, the Form I polymorph is defined as having an FT-Ramanspectrum comprising an absorption band at about 1633 cm⁻¹.

In some embodiments, the Form I polymorph is defined as having a powderX-ray diffraction spectrum comprising a peak at 5.0 (±0.2) degrees 2Θ.

In some embodiments, the Form I polymorph is defined as having anattenuated total reflection infrared spectrum comprising an absorptionband at one or more frequencies selected from the group consisting ofabout 1711, about 1682, about 1635, about 1599, about 1404, about 1182,and about 783 cm⁻¹. In some such embodiments, e.g., the Form I polymorphis defined as having an attenuated total reflection infrared spectrumcomprising an absorption band at one or more frequencies selected fromthe group consisting of about 1711 and about 1682 cm⁻¹. In other suchembodiments, the Form I polymorph is defined as having an attenuatedtotal reflection infrared spectrum comprising an absorption band at oneor more frequencies selected from the group consisting of about 1635,about 1404, and about 1182 cm⁻¹.

In some embodiments, the Form I polymorph is defined as having one (andtypically two or all three) of the following characteristics:

-   -   a. a powder X-ray diffraction spectrum substantially as shown in        FIG. 1,    -   b. an attenuated FT-Raman spectrum substantially as shown in        FIG. 2, or    -   c. an attenuated total reflection infrared spectrum        substantially as shown in FIG. 6.

Some embodiments are directed to compositions comprising20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, wherein at least adetectable amount of the 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolidein the composition is the Form I polymorph. In some such embodiments,e.g., at least about 50% (or at least about 75%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, or at leastabout 99.9%) of the 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide inthe composition is the Form I polymorph. In other such embodiments, atherapeutically effective amount of the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in the composition is theForm I polymorph. In still other such embodiments, the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in the composition issubstantially phase-pure Form I crystalline20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

In other embodiments, the invention is directed to the Form II polymorphof 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. Methods for makingthe Form II polymorph include, e.g., the method shown in Example 4. Aswith the Form I polymorph, the Form II polymorph tends to exhibit lesswater uptake than other solid-state forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide under, e.g., ambientconditions. It is hypothesized that the Form II polymorph exhibitsadvantageous physical stability, chemical stability, packing properties,thermodynamic properties, kinetic properties, surface properties,mechanical properties, filtration properties, or chemical purityrelative to other solid-state forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. The Form II polymorphalso is useful as an intermediate for preparing various othersolid-state forms. Table 1 summarizes examples of such methods.

TABLE 1 Use of the Form II Polymorph to Make Other Crystalline Forms of20,23-Dipiperidinyl-5-O-Mycaminosyl-Tylonolide Crystalline formIllustrations made from Form Example of method that may be of example IIpolymorph used method Form I polymorph Dissolve the Form II polymorph inExamples 12, a tBME/heptane solvent, and 13, and 16 remove the solventForm III polymorph Dissolve the Form II polymorph in Example 11acetonitrile solvent, subject the resulting mixture to repeated heatingand cooling cycles, and remove the solvent Ethyl acetate S1 Dissolve theForm II polymorph in Examples 6, crystalline solvate ethyl acetatesolvent, and remove 8, and 9 the solvent Ethanol S1 Dissolve the Form IIpolymorph in Example 17 crystalline solvate ethanol solvent, and removethe solvent Diethyl ketone S1 Dissolve the Form II polymorph in Example18 crystalline solvate diethyl ketone solvent, and remove the solventtBME S2 crystalline Dissolve the Form II polymorph in Example 19 solvatetBME solvent, and remove the solvent THF S3 crystalline Dissolve theForm II polymorph in Example 20 solvate THF solvent, and remove thesolvent Methyl acetate S4 Dissolve the Form II polymorph in Example 21crystalline solvate methyl acetate solvent, and remove the solvent Ethylformate S4 Dissolve the Form II polymorph in Example 22 crystallinesolvate ethyl formate solvent, and remove the solvent

The Form II polymorph may be identified using various analyticaltechniques. In some embodiments, the Form II polymorph is defined ashaving one (and typically two, three, four, or all five) of thefollowing characteristics:

-   -   a. an FT-Raman spectrum comprising an absorption band at one or        more frequencies selected from the group consisting of about        2929, about 1625, about 1595, about 1685, and 783 cm⁻¹;    -   b. a powder X-ray diffraction spectrum comprising a peak at 6.5        (±0.2) degrees 2Θ;    -   c. an attenuated total reflection infrared spectrum comprising        an absorption band at one or more frequencies selected from the        group consisting of about 2935, about 1736, about 1668, about        1587, about 1451, about 1165, about 1080, about 1057, about        1042, about 1005, about 981, about 838, and about 755 cm⁻¹;    -   d. a melting point of from about 113 to about 119° C.; or    -   e. a melting enthalpy of about 15 J/g.

In some embodiments, the Form II polymorph is defined as having anFT-Raman spectrum comprising an absorption band at about 2929 cm⁻¹. Inother embodiments, the Form II polymorph is defined as having anFT-Raman spectrum comprising an absorption band at about 1685 cm⁻¹.

In some embodiments, the Form II polymorph is defined as having a powderX-ray diffraction spectrum comprising a peak at 6.5 (±0.2) degrees 2Θ.

In some embodiments, the Form II polymorph is defined as having one (andtypically two or all three) of the following characteristics:

-   -   a. a powder X-ray diffraction spectrum substantially as shown in        FIG. 8,    -   b. an FT-Raman spectrum substantially as shown in FIG. 9, or    -   c. an attenuated total reflection infrared spectrum        substantially as shown in FIG. 13.

Some embodiments hereof are directed to compositions comprising20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide wherein at least adetectable amount of the 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolidein the composition is the Form II polymorph. In some such embodiments,e.g., at least about 50% (or at least about 75%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, or at leastabout 99.9%) of the 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide inthe composition is the Form II polymorph. In other such embodiments, atherapeutically effective amount of the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in the composition is theForm II polymorph. In still other such embodiments, the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in the composition issubstantially phase-pure Form II crystalline20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

In other embodiments, the disclosure is directed to the Form IIIpolymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.Illustrative methods for making the Form III polymorph include, e.g.,those shown in Examples 7, 10, and 11. It is believed that the Form IIIpolymorph exhibits greater stability relative to other solid-state formsof 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. It also ishypothesized that the Form III polymorph exhibits advantageous packingproperties, thermodynamic properties, kinetic properties, surfaceproperties, mechanical properties, filtration properties, or chemicalpurity relative to other solid-state forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. The Form III polymorphalso is, e.g., useful for making the Form I polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. This may be achieved by,e.g., dissolving Form III polymorph crystals in a tBME/heptane solvent,and removing the solvent. See, e.g., Example 15.

The Form III polymorph may be identified using various analyticaltechniques. In some embodiments, the Form III polymorph is defined ashaving one (and typically two, three, four, or all five) of thefollowing characteristics:

-   -   a. an FT-Raman spectrum comprising an absorption band at one or        more frequencies selected from the group consisting of about        2943, about 2917, about 1627, about 1590, about 1733, about        1669, about 1193, about 1094, and about 981 cm⁻¹;    -   b. a powder X-ray diffraction spectrum comprising at least one        peak selected from the group consisting of 5.6 (±0.2) and 6.1        (±0.2) degrees 2Θ;    -   c. an attenuated total reflection infrared spectrum comprising        an absorption band at one or more frequencies selected from the        group consisting of about 2931, about 1732, about 1667, about        1590, about 1453, about 1165, about 1081, about 1057, about        1046, about 1005, about 981, about 834, and about 756 cm⁻¹;    -   d. a melting point of from about 107 to about 134° C.; or    -   e. a melting enthalpy of about 38 J/g.

In some embodiments, the Form III polymorph is defined as having anFT-Raman spectrum comprising an absorption band at one or morefrequencies selected from the group consisting of about 2943, about2917, about 1590, about 1733, about 1669, about 1193, about 1094, andabout 981 cm⁻¹. In some such embodiments, e.g., the Form III polymorphis defined as having an FT-Raman spectrum comprising an absorption bandat one or more frequencies selected from the group consisting of about2943, about 2917, about 1590, about 1733, about 1094, and about 981cm⁻¹.

In some embodiments, the Form III polymorph is defined as having apowder X-ray diffraction spectrum comprising a peak at 6.1 (±0.2)degrees 2Θ.

In some embodiments, the Form III polymorph is defined as having one(and typically two or all three) of the following characteristics:

-   -   a. a powder X-ray diffraction spectrum substantially as shown in        FIG. 15,    -   b. an FT-Raman spectrum substantially as shown in FIG. 16, or    -   c. an attenuated total reflection infrared spectrum        substantially as shown in FIG. 20.

Some embodiments are directed to compositions comprising20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide wherein at least adetectable amount of the 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolidein the composition is the Form III polymorph. In some such embodiments,e.g., at least about 50% (or at least about 75%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, or at leastabout 99.9%) of the 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide inthe composition is the Form III polymorph. In other such embodiments, atherapeutically effective amount of the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in the composition is theForm III polymorph. In still other such embodiments, the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in the composition issubstantially phase-pure Form III crystalline20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

In other embodiments, the invention is directed to the Form IV polymorphof 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. Methods for makingthe Form IV polymorph include, e.g., the method shown in Example 23. Itis hypothesized that the Form IV polymorph exhibits advantageousphysical stability, chemical stability, packing properties,thermodynamic properties, kinetic properties, surface properties,mechanical properties, filtration properties, or chemical purityrelative to other solid-state forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

The Form IV polymorph may be identified using various analyticaltechniques. In some embodiments, the Form IV polymorph is defined ashaving one (and typically both) of the following characteristics:

-   -   a. an attenuated total reflection infrared spectrum comprising        an absorption band at one or more frequencies selected from the        group consisting of about 3559, about 2933, about 1743, about        1668, about 1584, about 1448, about 1165, about 1075, about        1060, about 1045, about 1010, about 985, about 839, and about        757 cm⁻¹; or    -   b. a melting point of from about 149 to about 155° C.

In some embodiments, the Form IV polymorph is defined as having anattenuated total reflection infrared spectrum having an absorption bandat 1743 cm⁻¹. In other embodiments, the Form IV polymorph is defined ashaving an attenuated total reflection infrared spectrum comprising anabsorption band at 3559 cm⁻¹.

In other embodiments, the Form IV polymorph is defined as having one(and typically both) of the following characteristics:

-   -   a. a powder X-ray diffraction spectrum substantially as shown in        FIG. 22, or    -   b. an attenuated total reflection infrared spectrum        substantially as shown in FIG. 24.

Some embodiments are directed to compositions comprising20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide wherein at least adetectable amount of the 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolidein the composition is the Form IV polymorph. In some such embodiments,e.g., at least about 50% (or at least about 75%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, or at leastabout 99.9%) of the 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide inthe composition is the Form IV polymorph. In other such embodiments, atherapeutically effective amount of the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in the composition is theForm IV polymorph. In still other such embodiments, the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in the composition issubstantially phase-pure Form IV crystalline20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

In other embodiments, the crystalline form of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide comprises a solvatedcrystalline form. In some embodiments, the solvated crystalline forms ofparticular interest are those that can be converted into a moredesirable solid-state form. In other embodiments, pharmaceuticallyacceptable solvated crystalline forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide are used directly inpharmaceutical compositions. It is hypothesized, e.g., that somecrystalline solvates tend to exhibit advantageous physical stability,chemical stability, packing properties, thermodynamic properties,kinetic properties, surface properties, mechanical properties,filtration properties, or chemical purity relative to other solid-stateforms of 20,23-piperidinyl-5-O-mycaminosyl-tylonolide. It also isbelieved that the solvated crystalline forms collectively can offer arange of different dissolution rates in, e.g., solid dosage forms. Whenused directly in pharmaceutical compositions, the solvated crystallineforms preferably are substantially exclusive of solvents that are notpharmaceutically acceptable.

In some embodiments, the crystalline form of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide comprises the S1crystalline solvate of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.Illustrative methods for making the ethyl acetate S1 crystalline solvateinclude, e.g., those shown in Examples 3 (Part E), 6, 8, and 9. Methodsfor making the ethanol S1 crystalline solvate include, e.g., the methodshown in Example 17. And methods for making the diethyl ketone S1crystalline solvate include, e.g., the method shown in Example 18. Theethyl acetate S1 crystalline solvate, e.g., is useful as an intermediatefor preparing other solid-state forms. Table 2 summarizes examples ofsuch methods.

TABLE 2 Use of Ethyl Acetate Crystalline Solvate to Make OtherCrystalline Forms of 20,23-Dipiperidinyl-5-O-Mycaminosyl-TylonolideCrystalline form Illustrations made from S1 Example of method that maybe of example solvate used method Form I Combine ethyl acetate S1solvate Example 3, polymorph crystals with heptane, heat the Part Fresulting mixture, and remove the heptane Form III Dry ethyl acetate S1solvate Examples 7 polymorph crystals under vacuum and 10 Form IVCombine ethyl acetate S1 crystals Example 23 polymorph with heptane;heat the resulting mixture to at least, e.g., about 80° C. for anextended period while stirring; and remove the heptane

In some embodiments, the crystalline form of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide comprises the S2crystalline solvate of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.Methods for making the S2 crystalline solvate include, e.g., the methodshown in Example 19. It is contemplated that the S2 crystalline solvate(i.e., the tBME solvated crystalline form) may be particularly suitablefor use directly in pharmaceutical compositions. This crystallinesolvate exhibits stability at, e.g., 60° C. at 1 mbar (absolute) for 1day.

In some embodiments, the crystalline form of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide comprises the S3crystalline solvate of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.Methods for making the S3 crystalline solvate include, e.g., the methodshown in Example 20.

In some embodiments, the crystalline form of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide comprises the S4crystalline solvate of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.Methods for making the methyl acetate S4 crystalline solvate include,e.g., the method shown in Example 21. And methods for making the ethylformate S4 crystalline solvate include, e.g., the method shown inExample 22.

Some embodiments are directed to compositions comprising20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide wherein at least adetectable amount of the 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolidein the composition is one of the above-referenced crystalline solvateforms. In some embodiments, e.g., at least about 50% (or at least about75%, at least about 85%, at least about 90%, at least about 95%, atleast about 99%, or at least about 99.9%) of the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in the composition is thecrystalline solvate form. In some such embodiments, at least about 50%(or at least about 75%, at least about 85%, at least about 90%, at leastabout 95%, at least about 99%, or at least about 99.9%) of the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in the composition is theethyl acetate 51 crystalline solvate. In other embodiments, atherapeutically effective amount of the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in the composition is inone of the above-listed crystalline solvate forms. In still otherembodiments, the 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in thecomposition is substantially phase-pure as to one of the above-discussedcrystalline solvate forms. In some such embodiments, e.g., the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in the composition issubstantially phase-pure ethyl acetate 51 crystalline solvate.

Some embodiments are directed to a combination of two or moresolid-state forms selected from the group consisting of the Form Ipolymorph, Form II polymorph, Form III polymorph, Form IV polymorph, andsolvated crystalline forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. Such combinations may beuseful in, e.g., the preparation of solid pharmaceutical compositionshaving a variety of dissolution profiles, including controlled-releasecompositions. In one embodiment, a combination comprises the Form Ipolymorph in at least a detectable amount, with the remaining20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide being one or moresolid-state forms selected from the group consisting of the Form IIpolymorph, Form III polymorph, Form IV polymorph, and solvatedcrystalline forms. In another embodiment, the combination comprises theForm II polymorph in at least a detectable amount, with the remaining20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide being one or moresolid-state forms selected from the group consisting of the Form Ipolymorph, Form III polymorph, Form IV polymorph, and solvatedcrystalline forms. In another embodiment, the combination comprises theForm III polymorph in at least a detectable amount, with the remaining20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide being one or moresolid-state forms selected from the group consisting of the Form Ipolymorph, Form II polymorph, Form IV polymorph, and solvatedcrystalline forms. In still another embodiment, the combinationcomprises the Form IV polymorph in at least a detectable amount, withthe remaining 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide being oneor more solid-state forms selected from the group consisting of the FormI polymorph, Form II polymorph, Form III polymorph, and solvatedcrystalline forms.

Depending on the intended use of the solid-state form of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, processingconsiderations may favor selection of a specific solid-state form or aspecific combination of such solid-state forms. The ease of preparingsolid-state forms of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (orsolid-state forms of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolidehaving a minimum phase purity) generally differs from one solid-stateform to another.

Characterization of Solid-State Forms Techniques

Samples of the solid-state forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide prepared in accordancewith this invention have been characterized using several differenttechniques. These techniques include the following.

Powder X-Ray diffraction (“PXRD”) spectra for all samples but the FormIV polymorph were obtained with a Bruker D8 Advance X-ray diffractometerusing Cu Kα radiation (wavelength for calculating d values: λ=1.5418 Å);35 kV/45 mA tube power; a VANTEC1 detector; and a 0.017° 2Θ step size,105±5 sec per step, and 2°-50° 2Θ scanning range. Silicon single-crystalsample holders having a 12 mm diameter and a 0.1 mm depth were used. ThePXRD spectrum for the Form IV polymorph was obtained with a SiemensD5000 x-ray diffractometer using Diffract Plus software, a 0.04° 2Θ stepsize, a 2 sec step time, a 5.0°-80.0 2Θ scanning range, the divergenceslit set at V20, the anti-scatter slit set at V20, the detector slitout, rotation, 40 kV generator tension, 30 mA generator current, a highsensitivity scintillation counter, and a Cu x-ray tube.

Fourier-transform Raman (“FT-Raman”) spectra were obtained with a BrukerRFS100 FT-Raman spectrometer with an Nd:YAG laser using 1064 nmexcitation wavelength, 100 mW laser power, a Ge-detector, 64 scans, arange of 50-3500 cm⁻¹, a 2 cm⁻¹ resolution, and an aluminum sampleholder.

Measurements of thermogravimetry coupled to Fourier transform infraredspectroscopy (“TG-FTIR”) were obtained using a NetzschThermo-Microbalance TG 209 with a Bruker Vector 22 FT-IR Spectrometerusing an aluminum crucible (with micro-hole or open), N₂ atmosphere,heating rate of 10° C./min, and temperature range of 25-250° C.

Thermogravimetry (“TG”) measurements were obtained with a Perkin ElmerTGS 2 thermogravimetric analyzer using an aluminum crucible (open), N₂atmosphere, heating rate of 10° C./min, and temperature range of 25-500°C.

Differential scanning calorimetry (“DSC”) measurements were obtained forthe Form I, II, and III polymorphs with a Perkin Elmer DSC 7differential scanning calorimeter using gold crucibles; a heating rateof 10° C./min. These measurements were performed in hermetically sealedsample pans closed under inert gas (i.e., in the absence of oxygen)after removal of any residual solvent and moisture. One scan wasperformed with the Form I polymorph. That scan was performed from −50°C. to about 210° C. Two scans were performed for the Form II and IIIpolymorphs, with the first scan being performed from −50° C. to 150° C.,and the second scan being performed from −50° C. to 200° C. DSCmeasurements were obtained for the Form IV polymorph with a MettlerDSC-822e using an aluminum crucible, air as the cover gas, a 10K/minheating rate, a heating range of 30 to 200° C., and a 5 mg sample size.Applicants believe DSC is particularly prone to variations, and should,therefore, be used cautiously.

Dynamic vapor sorption (“DVS”) measurements were obtained with a ProjektMesstechnik SPS11-100n water vapor sorption analyzer. The samples wereplaced into aluminum crucibles on a microbalance, and allowed toequilibrate at 25° C. and 50% relative humidity before initiating thefollowing pre-defined humidity program at 25° C.: 50-95-0-50% relativehumidity, and scanning with a 5% change in relative humidity per hourand with iso-humid equilibration periods at extreme values.

Infrared (“IR”) spectra were obtained using an Excalibur FT-IRspectrometer from Portmann Instruments AG (now Varian). Two techniqueswere used. The first technique was attenuated total reflection (“ATR”)infrared spectroscopy. To obtain a spectrum using ATR, a spatula tip ofsample was placed onto the sample area of the ATR cell (torque 120n*cm), and the infrared spectra were recorded from 3600 to 700 cm⁻¹. Thesecond technique used a sample mulled in nujol (i.e., a nujolsuspension). To obtain a spectrum using such a sample, a spatula tip ofsample was ground thoroughly in a motar with two or three drops of nujoluntil a homogenous paste was obtained. The paste, in turn, was spread ona NaCl plate, and pressed with a second NaCl plate to form a thinhomogenous film. For these samples, the infrared spectra were recordedfrom 3600 to 600 cm⁻¹.

Finally, Applicants made various observations regarding the shapes andsizes of the 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide solid-stateforms, and have summarized those observations below. Applicants note,however, that this information should be used with caution because othershapes and/or sizes of the crystalline forms might exist, depending onthe procedure used to make the solid-state forms.

Form I Polymorph

The following discussion provides various observed characteristics ofthe Form I polymorph.

i. Appearance of the Form I Polymorph

The Form I polymorph was generally in the form of small particles.

ii. Powder X-Ray Diffraction Spectrum for the Form I Polymorph

The observed PXRD spectrum for the Form I polymorph is shown in FIG. 1,and the corresponding data is shown in the following Table 3:

TABLE 3 X-Ray Diffraction Data for the Form I Polymorph Angle (2-Θdegrees) d Value (Å) Intensity (cps) Rel. Intensity (%) 5.0 17.673 3436.0 5.6 15.781 84 87.8 9.0 9.826 50 52.2 10.5 8.425 26 26.7 11.2 7.90014 14.3 12.6 7.025 59 62.3 13.5 6.559 35 37.0 13.7 6.463 58 60.7 14.46.151 36 37.7 14.6 6.067 49 51.3 15.5 5.717 38 39.3 15.8 5.609 21 21.716.1 5.505 62 65.0 16.4 5.405 48 50.6 16.6 5.340 31 32.0 16.8 5.277 95100.0 17.8 4.983 83 86.9 18.1 4.901 94 98.3 18.3 4.848 61 63.6 19.34.599 29 30.6 19.6 4.529 75 78.3 20.3 4.375 51 53.8 20.6 4.311 36 38.221.1 4.210 22 23.1 21.6 4.114 43 45.1 22.5 3.952 30 31.5 23.1 3.850 1515.9 24.3 3.663 24 25.2 24.8 3.590 19 19.8 25.1 3.548 18 19.2 26.5 3.36314 14.5 28.1 3.175 15 16.2 31.7 2.823 12 12.3 Characteristic features ofthe spectrum include initial peaks at 2Θ = 5.0° and 5.6°.

With some samples, the PXRD spectrum showed contamination to some extentwith amorphous 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. It isbelieved, however, that there was essentially no such amorphous materialin the sample corresponding to the above-discussed PXRD spectrum.

iii. FT-Raman Spectrum for the Form I Polymorph

The observed FT-Raman spectrum for the Form I polymorph is shown in FIG.2, and the corresponding data is shown in the following Table 4:

TABLE 4 FT-Raman Data for the Form I Polymorph Position (cm⁻¹) Intensity2935 0.234 2894 0.122 2788 0.038 1712 0.013 1683 0.022 1633 0.182 15960.330 1452 0.058 1394 0.023 1318 0.042 1295 0.036 1249 0.034 1206 0.0271156 0.020 1118 0.029 1041 0.035 975 0.026 887 0.023 864 0.023 844 0.022781 0.036 512 0.023 464 0.018 440 0.020 410 0.022 86 0.080Characteristic features of the spectrum include intense peaks at 2935cm⁻¹, 1633 cm⁻¹, and 1596 cm⁻¹; and smaller peaks at 1712 cm⁻¹, 1683cm⁻¹, and 781 cm⁻¹.

iv. Thermogravimetry for the Form I Polymorph

FIG. 3 shows the results from a TG-FTIR analysis of the Form Ipolymorph. A weight loss of 0.6% was observed in the temperature rangeof from 60 to 180° C. Applicants believe this is attributable to waterloss. Due to the small amount, Applicants further believe that thiswater loss resulted from surface-absorbed water rather than beingattributable to a hydrate.

v. Differential Scanning calorimetry for the Form I Polymorph

FIG. 4 shows the results from a DSC analysis for the Form I polymorph.There is a sharp melting peak at 195° C. with a melting enthalpy ofΔH_(fus) of 57 J/g. These are both greater than the melting points andmelting enthalpies for the Form II and Form III polymorphs. In FIG. 4,the T_(g) step is barely discernable. It is believed that this confirmsthat the sample was greater than 90% crystalline.

Samples of the Form I polymorph were independently analyzed to determinethe melting point. Samples having a purity of about 98% (w/w) exhibiteda melting point of 192 to 195° C.

vi. Dynamic Vapor Sorption for the Form I Polymorph

FIG. 5 shows the results for a DVS analysis of the Form I polymorph,which was conducted at 25° C. A maximum water uptake less than 1% (w/w)at 95% relative humidity was observed.

vii. IR Spectrum for the Form I Polymorph

FIG. 6 shows ATR-IR spectrum for the Form I polymorph, and FIG. 7provides the IR spectrum for the Form I polymorph in a nujol suspension.The corresponding data are shown in the following Table 5:

TABLE 5 IR Data for the Form I Polymorph Frequencies of intense IRFrequencies of intense IR absorption bands observed with absorptionbands observed with ATR technique (cm⁻¹) Nujol technique (cm⁻¹) 35442932 overlaps with nujol vibration band 1711 1712 1682 1683 1635 16351599 1599 1442 overlaps with nujol vibration band 1404 1406 1373overlaps with nujol vibration band 1350 1351 1307 1309 1262 1263 11821187 1123 1107 1108 1079 1082 1053 1054 1008 1009 985 986 958 960 928909 900 877 861 842 843 818 816 783 782 722 Characteristic features ofthe spectra, particularly the ATR spectrum, include intense absorptionbands at 2932 cm⁻¹, 1711 cm⁻¹, 1682 cm⁻¹, 1599 cm⁻¹, 1442 cm⁻¹, 1182cm⁻¹, 1079 cm⁻¹, 1053 cm⁻¹, 1008 cm⁻¹, 985 cm⁻¹, 842 cm⁻¹, and 783 cm⁻¹.Absorption bands at 1711 cm⁻¹ and 1682 cm⁻¹ appear to be particularlyunique to this polymorph. Absorption bands at 1635 cm⁻¹, 1404 cm⁻¹, and1182 cm⁻¹ also appear to be particularly unique to this polymorph.

Form II Polymorph

The following discussion provides various observed characteristics ofthe Form II polymorph.

i. Appearance of the Form II Polymorph

The Form II polymorph was generally in the form of prismatic crystalswith a size of up to several hundred microns.

ii. Powder X-Ray Diffraction Spectrum for the Form II Polymorph

The observed PXRD spectrum for the Form II polymorph is shown in FIG. 8,and the corresponding data is shown in the following Table 6:

TABLE 6 X-Ray Diffraction Data for the Form II Polymorph Angle (2-Θdegrees) d Value (Å) Intensity (cps) Rel. Intensity (%) 6.5 13.598250.71 100.0 8.7 10.164 86.09 34.3 9.7 9.118 29.90 11.9 9.9 8.934 35.3814.1 12.4 7.138 34.12 13.6 13.0 6.810 43.56 17.4 15.0 5.906 55.07 22.015.8 5.609 44.94 17.9 16.1 5.505 19.86 7.9 16.3 5.438 22.40 8.9 17.05.216 85.44 34.1 17.9 4.955 30.02 12.0 18.1 4.901 56.47 22.5 19.7 4.50650.08 20.0 20.0 4.439 125.24 50.0 21.3 4.171 43.10 17.2 24.9 3.576 14.936.0 26.3 3.389 12.77 5.1 27.4 3.255 15.40 6.1 Characteristic features ofthe spectrum include the initial and most intense peak being at 2Θ =6.5°.

iii. FT-Raman Spectrum for the Form II Polymorph

The observed FT-Raman spectrum for the Form II polymorph is shown inFIG. 9, and the corresponding data is shown in the following Table 7:

TABLE 7 FT-Raman Data for the Form II Polymorph Position (cm⁻¹)Intensity 2929 0.435 1685 0.044 1625 0.550 1595 1.118 1451 0.114 13610.062 1311 0.100 1270 0.085 1248 0.100 1195 0.074 1117 0.060 1095 0.0751023 0.073 984 0.047 925 0.051 873 0.058 783 0.084 513 0.063 379 0.06687 0.198 Characteristic features of the spectrum include intense peaksat 2929 cm⁻¹, 1625 cm⁻¹, and 1595 cm⁻¹; and smaller, but sharp, peaks at1685 cm⁻¹ and 783 cm⁻¹.

iv. Thermogravimetry for the Form II Polymorph

FIG. 10 shows the results from a TG-FTIR analysis of the Form IIpolymorph. A weight loss of 0.7% was observed, mainly in the temperaturerange of from 50 to 100° C. Applicants believe this is attributable towater loss. Decomposition began at a temperature of greater than 220° C.

v. Differential Scanning calorimetry for the Form II Polymorph

FIG. 11 shows the results from a DSC analysis of the Form II polymorph.The first scan (the continuous line) shows a melting peak at 113° C.with a melting enthalpy of ΔH_(fus)=15 J/g. The second scan (the dottedline) shows a glass transition temperature (“T_(g)”) of 96.1° C.Re-crystallization was not observed.

Samples of the Form II polymorph were independently analyzed todetermine the melting point. Samples having a purity of about 96% (w/w)exhibited a melting point of from 113 to 119° C.

vi. Dynamic Vapor Sorption for the Form II Polymorph

FIG. 12 shows the results from a DVS analysis of the Form II polymorph.This analysis was conducted at 25° C. A maximum water uptake of about 2%(by weight) at 95% relative humidity was observed.

vii. IR Spectrum for the Form II Polymorph

FIG. 13 shows ATR-IR spectrum for the Form II polymorph, and FIG. 14provides the IR spectrum for the Form II polymorph in a nujolsuspension. The corresponding data are shown in the following Table 8:

TABLE 8 IR Data for the Form II Polymorph Frequencies of intense IRFrequencies of intense IR absorption bands observed with absorptionbands observed with ATR technique (cm⁻¹) Nujol technique (cm⁻¹) 35402935 overlaps with nujol vibration band 1736 1741 1668 1669 1626 15871591 1451 overlaps with nujol vibration band 1372 overlaps with nujolvibration band 1352 1349 1310 1313 1302 1277 1277 1242 1245 1217 11871165 1166 1116 1080 1087 1057 1058 1042 1044 1005 1005 981 980 966 966934 933 910 908 882 859 858 838 837 811 781 780 755 755 722Characteristic features of the spectra, particularly the ATR spectrum,include intense absorption bands at 2935 cm⁻¹, 1736 cm⁻¹, 1668 cm⁻¹,1587 cm⁻¹, 1451 cm⁻¹, 1165 cm⁻¹, 1080 cm⁻¹, 1057 cm⁻¹, 1042 cm⁻¹, 1005cm⁻¹, 981 cm⁻¹, 838 cm⁻¹, and 755 cm⁻¹.

Form III Polymorph

The following observed characteristics of the Form III polymorph wereobserved.

i. Appearance of the Form III Polymorph

The Form III polymorph was generally in the form of fine needles.

ii. Powder X-Ray Diffraction Spectrum for the Form III Polymorph

The observed PXRD spectrum for the Form III polymorph is shown in FIG.15, and the corresponding data is shown in the following Table 9:

TABLE 9 X-Ray Diffraction Data for the Form III Polymorph Angle (2-Θdegrees) d Value (Å) Intensity (cps) Rel. Intensity (%) 5.6 15.781 3617.3 6.1 14.489 209 100.0 8.3 10.653 54 25.8 11.0 8.043 44 21.2 12.27.255 40 19.2 13.2 6.707 45 21.4 13.7 6.463 21 9.8 14.2 6.237 26 12.415.2 5.829 16 7.9 15.7 5.644 53 25.2 16.1 5.505 19 9.2 16.8 5.277 10550.1 17.9 4.955 28 13.3 18.2 4.874 22 10.6 18.9 4.695 37 17.8 19.6 4.52917 8.1 20.5 4.332 86 41.1 21.6 4.114 44 21.2 22.5 3.952 20 9.4 24.33.663 14 6.6 26.0 3.427 15 7.1 Characteristic features of the spectruminclude the most intense peak being at 2Θ = 6.1°, which is accompaniedby a smaller peak at 2Θ = 5.6°. It has been observed that the relativeintensity of these two peaks varies from batch to batch, as do therelative intensities of other peaks in the spectrum. Such variations arenot uncommon to PXRD. Often, they originate from orientation effects,particularly in the context of anisotropic (i.e., needle- andplate-like) crystals. These variations, however, generally do notinfluence the identification of the polymorphic form because thisnormally depends on peak positions rather than intensities.

iii. FT-Raman Spectrum for the Form III Polymorph

The observed FT-Raman spectrum for the Form III polymorph is shown inFIG. 16, and the corresponding data is shown in the following Table 10:

TABLE 10 FT-Raman Data for the Form III Polymorph Position (cm⁻¹)Intensity 2943 0.214 2917 0.199 2785 0.049 1733 0.014 1669 0.038 16270.256 1590 0.447 1443 0.064 1314 0.040 1269 0.051 1257 0.037 1217 0.0211193 0.037 1116 0.035 1094 0.043 1041 0.029 981 0.034 908 0.026 8810.031 863 0.025 833 0.022 810 0.018 781 0.027 505 0.030 444 0.021 3990.025 216 0.042 173 0.033 108 0.070 84 0.068 Characteristic features ofthe spectrum include intense peaks at 2943 cm⁻¹, 2917 cm⁻¹, 1627 cm⁻¹,and 1590 cm⁻¹; and smaller peaks at 1733 cm⁻¹, 1669 cm⁻¹, 1193 cm⁻¹,1094 cm⁻¹, and 981 cm⁻¹.

iv. Thermogravimetry for the Form III Polymorph

TG-FTIR analysis of one sample showed a weight loss of 1.7% up to 220°C., with most of the loss occurring between 50 and 120° C. It ishypothesized that this weight loss was due to water or acetonitrile inthe sample (the sensitivity of the IR-detector to acetonitrile is low).

FIG. 17 shows the results of a TG analysis of the Form III polymorph. Aweight loss of less than 0.05% was observed up to 200° C. Decompositionbegan at a temperature of greater than 270° C.

v. Differential Scanning calorimetry for the Form III Polymorph

FIG. 18 shows the results from a DSC analysis of the Form III polymorph.The first scan (the continuous line) shows a melting peak at 134° C.with a melting enthalpy of ΔH_(fus)=38 J/g. Upon cooling, the materialsolidified into the amorphous state. The second scan (the dotted line)shows a T_(g) of 96° C., re-crystallization, and melting again at 195°C.

Samples of the Form III polymorph were independently analyzed todetermine the melting point. Samples having a purity of about 99%exhibited a melting point of from 122 to 126° C.

vi. Dynamic Vapor Sorption for the Form III Polymorph

FIG. 19 shows the results of a DVS analysis of the Form III polymorph.This analysis was conducted at 25° C. A water uptake of about 6% wasobserved between 70-85% relative humidity.

vii. IR Spectrum for the Form III Polymorph

FIG. 20 shows ATR-IR spectrum for the Form III polymorph, and FIG. 21provides the IR spectrum of Form III polymorph in a nujol suspension.The corresponding data are shown in Table 11:

TABLE 11 IR Data for the Form III Polymorph Frequencies of intense IRFrequencies of intense IR absorption bands observed with absorptionbands observed with ATR technique (cm⁻¹) Nujol technique (cm⁻¹) 35412931 overlaps with nujol vibration band 1732 1734 1667 1669 1626 15901591 1453 overlaps with nujol vibration band 1376 overlaps with nujolvibration band 1350 1350 1304 1312 1277 1277 1256 1256 1217 1217 11891165 1165 1081 1087 1057 1060 1046 1005 1004 981 980 965 966 934 935 908908 881 859 834 836 812 811 780 756 757 722 Characteristic features ofthe spectra, particularly the ATR spectrum, include intense absorptionbands at 2931 cm⁻¹, 1732 cm⁻¹, 1667 cm⁻¹, 1590 cm⁻¹, 1453 cm⁻¹, 1165cm⁻¹, 1081 cm⁻¹, 1057 cm⁻¹, 1046 cm⁻¹, 1005 cm⁻¹, 981 cm⁻¹, 834 cm⁻¹,and 756 cm⁻¹.

Form IV Polymorph

The following observation of the Form IV polymorph were made.

i. Powder X-Ray Diffraction Spectrum for the Form IV Polymorph

The observed PXRD spectrum for the Form IV polymorph is shown in FIG.22, and the corresponding data is shown in the following Table 12:

TABLE 12 X-Ray Diffraction Data for the Form IV Polymorph Angle (2-Θdegrees) d Value (Å) Intensity (cps) Rel. Intensity (%) 6.232 14.171 1356.4 6.848 12.898 367 17.5 8.653 10.211 709 33.8 10.16 8.7007 1650 78.710.64 8.3119 887 42.3 11.18 7.9106 706 33.7 11.95 7.4034 314 15.0 12.297.1957 253 12.1 13.84 6.3955 328 15.6 15.06 5.8779 804 38.3 15.49 5.7158831 39.6 16.06 5.5147 1600 76.3 17.50 5.0638 2097 100 18.75 4.7291 114454.6 19.93 4.4510 1435 68.4 20.49 4.3321 1815 86.6 21.40 4.1473 118956.7 22.59 3.9328 838 40.0 23.50 3.7830 517 24.7 23.96 3.7118 745 35.525.06 3.5507 493 23.5 25.32 3.5147 522 24.9 25.74 3.4588 574 27.4 26.923.3091 464 22.1 27.83 3.2036 691 33.0 28.24 3.1576 470 22.4 29.02 3.0748389 18.6 29.36 3.0396 335 16.0 30.92 2.8902 454 21.6 31.30 2.8554 50624.1 32.42 2.7594 650 31.0 33.60 2.6649 361 17.2 35.38 2.5347 436 20.835.85 2.5027 390 18.6 36.66 2.4492 345 16.5 37.66 2.3869 426 20.3 38.632.3287 426 20.3

ii. Differential Scanning calorimetry for the Form IV Polymorph

FIG. 23 shows the results from a DSC analysis of the Form IV polymorph.The curve shows a peak at 155° C., believed to correspond to the Form IVpolymorph. The curve also shows a peak at 191° C., believed tocorrespond to the Form I polymorph. It is believed that the samplecontained both of the Form I and Form IV polymorphs, or that the Form IVpolymorph converts to the Form I polymorph during heating. Samples ofthe Form IV polymorph were independently analyzed to determine themelting point. Samples being about 90.0% (w/w) pure exhibited meltingpoints of from 149-152° C.

iv. IR Spectrum for the Form IV Polymorph

FIG. 24 shows ATR-IR spectrum for the Form IV polymorph, and FIG. 25provides the IR spectrum for the Form IV polymorph in a nujolsuspension. The corresponding data are shown here:

TABLE 13 IR Data for the Form IV Polymorph Frequencies of intense IRFrequencies of intense IR absorption bands observed with absorptionbands observed with ATR technique (cm⁻¹) Nujol technique (cm⁻¹) 35593568 2933 overlaps with nujol vibration band 1743 1745 1668 1670 16201584 1588 1448 overlaps with nujol vibration band 1441 1394 1370overlaps with nujol vibration band 1351 1314 1307 1272 1271 1259 12591215 1195 1195 1165 1166 1140 1118 1116 1075 1075 1060 1045 1046 10101010 985 992 954 953 936 910 872 860 839 839 810 785 784 757 756 722Characteristic features of the spectra, particularly the ATR spectrum,include intense absorption bands at 2933 cm⁻¹, 1743 cm⁻¹, 1668 cm⁻¹,1584 cm⁻¹, 1448 cm⁻¹, 1165 cm⁻¹, 1075 cm⁻¹, 1060 cm⁻¹, 1045 cm⁻¹, 1010cm⁻¹, 985 cm⁻¹, 839 cm⁻¹, and 757 cm⁻¹. The absorption band at 3559 cm⁻¹appears to be particularly unique to this polymorph.

S1 Crystalline Solvate

The following discussion provides various observed characteristics ofthe S1 crystalline solvate. Although the PXRD and FT-Raman data belowcorrespond to the ethyl acetate S1 crystalline solvate, this data isgenerally applicable to characterizing the diethyl ketone and ethanolcrystalline solvates as well because they are isomorphic with the ethylacetate crystalline solvate.

i. Appearance of the Ethyl Acetate S1 crystalline solvate

The ethyl acetate S1 crystalline solvate was generally in the form offine needles, or larger crystals with a tendency to break down intofibers.

ii. Powder X-Ray Diffraction Spectrum for the Ethyl Acetate S1Crystalline Solvate

The observed PXRD spectrum for the ethyl acetate S1 crystalline solvateis shown in FIG. 26, and the corresponding data is shown in thefollowing Table 14:

TABLE 14 X-Ray Diffraction Data for the Ethyl Acetate S1 CrystallineSolvate Angle (2-Θ degrees) d Value (Å) Intensity (cps) Rel. Intensity(%) 5.6 15.781 199 100.0 6.1 14.489 62 31.3 7.1 12.450 32 15.9 8.310.653 30 15.2 11.0 8.043 32 15.9 11.2 7.900 62 31.3 11.5 7.695 37 18.712.0 7.375 22 10.9 12.2 7.255 17 8.5 12.8 6.916 17 8.5 13.3 6.657 2412.2 13.5 6.559 47 23.6 13.8 6.417 31 15.6 14.4 6.151 26 13.3 14.9 5.94668 34.3 15.7 5.644 18 8.9 16.8 5.277 71 36.0 17.2 5.155 27 13.6 17.84.983 89 44.8 19.0 4.671 41 20.8 19.4 4.575 23 11.5 20.3 4.375 29 14.820.5 4.332 29 14.7 21.6 4.114 22 11.0 22.1 4.022 19 9.7 22.6 3.934 2110.7 23.9 3.723 17 8.6 25.2 3.534 13 6.5 Characteristic features of thespectrum include initial peaks at 2Θ = 5.6° and 6.1°.

iii. FT-Raman Spectrum for the Ethyl Acetate S1 Crystalline Solvate

The observed FT-Raman spectrum for the ethyl acetate S1 crystallinesolvate is shown in FIG. 27, and the corresponding data is shown in thefollowing Table 15:

TABLE 15 FT-Raman Data for the Ethyl Acetate S1 Crystalline SolvatePosition (cm⁻¹) Intensity 2936 0.212 2877 0.098 2857 0.078 2775 0.0401745 0.012 1669 0.029 1625 0.198 1586 0.363 1451 0.058 1392 0.022 13000.033 1271 0.039 1244 0.030 1215 0.021 1193 0.029 1118 0.028 1097 0.0371040 0.034 1008 0.024 978 0.038 911 0.018 882 0.024 833 0.018 812 0.017781 0.023 554 0.012 504 0.029 445 0.019 398 0.021 84 0.076Characteristic features of the spectrum include intense peaks at 2936cm⁻¹, 1625-1627 cm⁻¹, and 1586 cm⁻¹; and smaller, but sharp, peaks at1745 cm⁻¹, 1669 cm⁻¹, and 978 cm⁻¹.

iv. Thermogravimetry for S1 Crystalline Solvates

FIGS. 28, 29, and 30 show the TG-FTIR results for the ethyl acetate,ethanol, and diethyl ketone S1 crystalline solvates, respectively. Theseresults confirm the existence of crystalline solvates with approximatelyone solvent molecule per 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolidemolecule, assuming essentially pure20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. The ethyl acetate S1crystalline solvate exhibited a weight loss of approximately 4.1%resulting from the liberation of ethyl acetate. This corresponds to amole ratio of ethyl acetate to20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide of 0.36. The ethanol S1crystalline solvate exhibited a weight loss of approximately 6.6% up to200° C. resulting from the liberation of ethanol (the S1 crystallinesolvate also may contain a small amount of water, which may have beenliberated as well). This corresponds to a mole ratio of ethanol to20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide of 1.1. And the diethylketone S1 crystalline solvate exhibited a weight loss 10% resulting fromthe liberation of diethyl ketone. This corresponds to a mole ratio ofdiethyl ketone to 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide of 1.0.These results are summarized in Table 16:

TABLE 16 TG-FTIR Results for S1 Crystalline Solvates Solvent moleculesper Crystalline Detected 20,23-dipiperidinyl-5-O- Solvate Solventsmycaminosyl-tylonolide molecule ethyl acetate ethyl acetate ~0.4 ethylacetate + water 0.6 water ethanol Ethanol 1.1 ethanol diethyl ketonediethyl ketone 1.0 diethyl ketoneFor each of the crystalline solvates, the weight loss begins at from 40to 50° C. This indicates relatively low stability, and is consistentwith the observation that the ethyl acetate crystalline solvate can,e.g., be readily converted into the Form III polymorph by vacuum dryingat ambient temperature. See, e.g., Example 7. The results for the ethylacetate crystalline solvate indicate that the solvent molecule can besubstituted by water, and is consistent with the DVS results for theForm III polymorph.

S2 Crystalline Solvate

The following discussion provides various observed characteristics ofthe S2 crystalline solvate.

i. Appearance of the tBME S2 Crystalline Solvate

The tBME S2 crystalline solvate was generally in the form of poorlydefined crystals, and did not exhibit a tendency to break down intofibers, as compared to the S1 solvate crystals.

ii. Solubility of the tBME S2 Crystalline Solvate in tBME

The solubility of the tBME S2 crystalline solvate in tBME is between 40and 50 mg/ml. Accordingly, the solubility is at least one order ofmagnitude less than the solubility of the Form II polymorph in tBME.

iii. Powder X-Ray Diffraction Spectrum for the tBME S2 CrystallineSolvate

The observed PXRD spectrum for the tBME S2 crystalline solvate is shownin FIG. 31, and the corresponding data is shown in the following Table17:

TABLE 17 X-Ray Diffraction Data for the tBME S2 Crystalline SolvateAngle (2-Θ degrees) d Value (Å) Intensity (cps) Rel. Intensity (%) 6.114.489 54 73.5 8.6 10.282 20 26.8 9.5 9.309 13 17.3 10.0 8.845 52 71.710.3 8.588 56 77.4 10.9 8.117 16 21.5 12.3 7.196 18 24.0 13.5 6.559 3953.7 13.8 6.417 20 27.1 14.4 6.151 24 33.4 14.7 6.026 23 32.0 15.6 5.68023 31.4 15.9 5.574 34 46.5 16.4 5.405 38 52.1 17.0 5.216 70 95.4 17.25.155 28 38.1 18.0 4.928 33 45.9 18.6 4.770 73 100.0 18.8 4.720 35 48.319.3 4.599 35 48.0 20.1 4.418 58 79.9 21.3 4.171 16 21.3 21.5 4.133 1621.4 22.3 3.986 15 20.3 22.6 3.934 13 18.1 23.0 3.867 13 18.1 23.3 3.81813 17.4 24.1 3.693 18 24.8 25.8 3.453 14 19.7 26.4 3.376 12 17.0 28.13.175 11 15.7 Characteristic features of the spectrum include severalpeaks with similar intensity at 2Θ = 6.1°, 10.0°, 10.3°, 17.0°, 18.6°,and 20.1°.

iv. FT-Raman Spectrum for the tBME S2 Crystalline Solvate

The observed FT-Raman spectrum for the tBME S2 crystalline solvate isshown in FIG. 32, and the corresponding data is shown in the followingTable 18:

TABLE 18 FT-Raman Data for the tBME S2 Crystalline Solvate Position(cm⁻¹) Intensity 2928 0.369 2883 0.184 1674 0.049 1623 0.353 1587 0.7011445 0.108 1393 0.039 1318 0.067 1296 0.056 1276 0.066 1244 0.089 11900.073 1154 0.034 1126 0.053 1069 0.051 1038 0.058 1007 0.040 979 0.044890 0.040 838 0.033 780 0.056 728 0.063 503 0.045 439 0.035 418 0.035 850.128 Characteristic features of the spectrum include intense peaks at2928 cm⁻¹, 1623 cm⁻¹, and 1587 cm⁻¹; and smaller, but sharp, peaks at1674 cm⁻¹, 1244 cm⁻¹, 1190 cm⁻¹, 780 cm⁻¹, and 728 cm⁻¹.

v. Thermogravimetry for the tBME S2 Crystalline Solvate

FIG. 33 shows the TG-FTIR results for a sample of the tBME S2crystalline solvate. A weight loss of from about 8.7-10% occurred due totBME liberation. This weight loss corresponds to about 0.8-0.9 tBMEmolecule per molecule of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide,assuming essentially pure20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. Almost all the weightloss occurs above 90° C., with a sharp step when the temperatureincreases to greater than 100° C. Thus, the majority of the weight lossoccurs at temperatures greater than the boiling point of tBME. The tBMEcrystalline solvate appears to be more stable than the ethyl acetate S1crystalline solvate. The stability was confirmed by a desolvationexperiment wherein no loss of solvent was observed upon drying undervacuum at both ambient temperature and 70° C.

S3 Crystalline Solvate

The following discussion provides various observed characteristics ofthe S3 crystalline solvate.

i. Appearance of the THF S3 Crystalline Solvate

The THF S3 crystalline solvate was generally in the form of irregularchunks, and did not exhibit a tendency to break down into fibers, ascompared to the S1 crystalline solvate crystals.

ii. Powder X-Ray Diffraction Spectrum for the THF S3 Crystalline Solvate

The observed PXRD spectrum for the THF S3 crystalline solvate is shownin FIG. 34, and the corresponding data is shown in the following Table19:

TABLE 19 X-Ray Diffraction Data for the THF S3 Crystalline Solvate Angle(2-Θ degrees) d Value (Å) Intensity (cps) Rel. Intensity (%) 6.2 14.25535 52.4 8.6 10.282 21 30.6 9.5 9.309 17 25.3 10.1 8.758 47 69.7 10.58.425 64 94.7 12.5 7.081 13 20.0 13.6 6.511 56 82.8 14.0 6.326 22 32.514.6 6.067 28 41.3 14.8 5.985 25 36.5 15.8 5.609 22 32.1 16.0 5.539 2841.7 16.7 5.309 46 67.7 17.2 5.155 59 88.1 17.4 5.096 35 52.6 17.7 5.01120 29.6 18.2 4.874 33 48.3 18.8 4.720 64 94.7 19.0 4.671 49 73.4 19.64.529 35 51.3 20.5 4.332 67 100.0 21.6 4.114 17 25.4 22.8 3.900 16 23.123.6 3.770 13 19.6 24.5 3.633 14 20.6 26.2 3.401 12 17.8 27.4 3.255 1015.4 Characteristic features of the spectrum include several peaks withsimilar intensity at 2Θ = 6.2°, 10.1°, 10.5°, 13.6°, 16.7°, 17.2°,18.8°, and 20.5°.

iii. FT-Raman Spectrum for the THF S3 Crystalline Solvate

The observed FT-Raman spectrum for the THF S3 crystalline solvate isshown in FIG. 35, and the corresponding data is shown in the followingTable 20

TABLE 20 FT-Raman Data for the THF S3 Crystalline Solvate Position(cm⁻¹) Intensity 2928 0.340 2883 0.192 1673 0.051 1622 0.405 1586 0.8281451 0.111 1394 0.039 1318 0.074 1296 0.066 1269 0.073 1244 0.092 11910.080 1127 0.057 1067 0.052 1040 0.066 980 0.051 910 0.052 890 0.045 8390.036 782 0.057 503 0.041 438 0.037 419 0.036 100 0.138 Characteristicfeatures of the spectrum include intense peaks at 2928 cm⁻¹, 1622 cm⁻¹,and 1586 cm⁻¹; and smaller, but sharp, peaks at 1673 cm⁻¹, 1244 cm⁻¹,1191 cm⁻¹, and 782 cm⁻¹.

iv. Thermogravimetry for the THF S3 Crystalline Solvate

FIG. 36 shows the TG-FTIR results for the THF S3 crystalline solvate.The majority of the weight loss occurred at temperatures that aregreater than the boiling point of THF. Specifically, less than 10% ofthe weight loss occurred at from 60 to 100° C., whereas approximately80% of the loss occurred at from 110 to 180° C. The results show aweight loss of about 8.1% occurring at temperatures greater than 100° C.due to liberation of THF. This corresponds to about 0.8 THF moleculesper molecule of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, assumingessentially pure 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

S4 Crystalline Solvate

The following discussion provides various observed characteristics ofthe S4 crystalline solvate. Although the PXRD and FT-Raman data belowcorrespond to the methyl acetate S4 crystalline solvate, this data isgenerally applicable to characterizing the ethyl formate crystallinesolvate as well because it is isomorphic with the methyl acetatecrystalline solvate.

i. Appearance of the Methyl Acetate S4 Crystalline Solvate

The methyl acetate S4 crystalline solvate contained some well-developedprismatic crystals. The crystals did not exhibit a tendency to breakdown into fibers, as compared to the S1 solvate crystals.

ii. Powder X-Ray Diffraction Spectrum for the Methyl Acetate S4Crystalline Solvate

The observed PXRD spectrum for the methyl acetate S4 crystalline solvateis shown in FIG. 37, and the corresponding data is shown in thefollowing Table 21:

TABLE 21 X-Ray Diffraction Data for the Methyl Acetate S4 CrystallineSolvate Angle (2-Θ degrees) d Value (Å) Intensity (cps) Rel. Intensity(%) 6.3 14.029 144 66.5 8.7 10.164 44 20.3 9.5 9.309 25 11.7 10.1 8.75888 40.6 10.5 8.425 90 41.3 11.0 8.043 44 20.2 11.7 7.563 24 11.2 12.67.025 44 20.3 13.1 6.758 26 11.9 13.6 6.511 54 25.1 13.9 6.371 40 18.514.1 6.281 49 22.4 14.8 5.985 120 55.3 15.8 5.609 50 23.2 16.8 5.277 10950.1 17.4 5.096 161 74.0 18.0 4.928 71 32.7 18.9 4.695 217 100.0 19.84.484 73 33.4 20.5 4.332 49 22.6 20.9 4.250 145 66.6 21.8 4.077 36 16.622.5 3.952 26 12.0 23.5 3.786 58 26.8 24.2 3.678 23 10.4 25.0 3.562 4118.8 26.6 3.351 34 15.7 27.8 3.209 23 10.4 28.5 3.132 29 13.4 29.7 3.00825 11.7 32.7 2.739 17 7.7 Characteristic features of the spectruminclude several peaks with similar intensity at 2Θ = 6.3°, 10.1°, 10.5°,14.8°, 16.8°, 17.4°, 18.9°, and 20.9°.

iii. FT-Raman Spectrum for the Methyl Acetate S4 Crystalline Solvate

The observed FT-Raman spectrum for the methyl acetate S4 crystallinesolvate is shown in FIG. 38, and the corresponding data is shown in thefollowing Table 22:

TABLE 22 FT-Raman Data for the Methyl Acetate S4 Crystalline SolvatePosition (cm⁻¹) Intensity 2949 0.205 2934 0.223 1740 0.019 1671 0.0341619 0.242 1581 0.468 1452 0.075 1394 0.027 1318 0.047 1296 0.045 12690.048 1243 0.058 1191 0.054 1155 0.024 1128 0.038 1091 0.037 1040 0.0481008 0.031 981 0.041 889 0.028 864 0.023 841 0.039 815 0.017 782 0.039641 0.021 502 0.026 436 0.027 419 0.024 92 0.100 Characteristic featuresof the spectrum include intense peaks at 2949 cm⁻¹, 2934 cm⁻¹, 1619-1621cm⁻¹, and 1581-1584 cm⁻¹; and smaller, but sharp, peaks at 1671 cm⁻¹,1243 cm⁻¹, 1191 cm⁻¹, 981 cm⁻¹, and 782 cm⁻¹.

iv. Thermogravimetry for S4 Crystalline Solvates

FIGS. 39 and 40 show the TG-FTIR results for the methyl acetate andethyl formate S4 crystalline solvates, respectively. These resultsconfirm the existence of the crystalline solvates. For the methylacetate crystalline solvate, there was a weight loss of about 8.4% dueto liberation of methyl acetate. And for the ethyl formate crystallinesolvate, there was a weight loss of about 7.7%. Based on the observedresults, it is estimated that the methyl acetate crystalline solvate hasa about 0.9 methyl acetate molecules per20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide molecule, and the ethylformate crystalline solvate has from about 0.6 to about 0.8 ethylformate molecules per 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolidemolecule. Both these estimates assume essentially pure20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. Less than 10% of theweight loss occurred at from 70 to 110° C. for the methyl acetatecrystalline solvate, and from 60 to 90° C. for the ethyl formatecrystalline solvate. For both crystalline solvates, the desolvationproceeded quickly. The desolvation was nearly completed at 160° C. forthe methyl acetate crystalline solvate, and 130° C. for the ethylformate crystalline solvate.

E. Preparation of Medicaments and Methods of Treatment Using Macrolides

The macrolides described above (including macrolides prepared by theprocess described above, as well as the various crystalline forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide described above) may beused, e.g., to treat pasteurellosis in animals, particularly livestockand poultry. In some embodiments, such a macrolide(s) is used to treatbovine animals having bovine respiratory disease (BRD) associated withMannheimia haemolytica, Pasteurella multocida and Histophilus somni. Inother embodiments, such a macrolide(s) is used to treat swine animalshaving swine respiratory disease associated with Actinobacilluspleuropneumoniae, Pasteurella multocida, and Bordetella bronchiseptica.

In general, a therapeutically-effective amount of one or more of suchmacrolides is administered to the recipient animal. As used in thisdisclosure, the term “therapeutically effective amount” constitutes anamount that is sufficient to prevent, reduce the risk of, delay theonset of, ameliorate, suppress, or eradicate a target pathogen(s)infection. Generally, the therapeutically effective amount is defined asthe amount necessary to achieve a concentration efficacious to controlthe target pathogen(s) at the site of infection (or, when used toprevent, reduce the risk of, or delay the onset of infection, at a sitesusceptible to infection). The concentration at the site of infection(or at a site susceptible to infection) is preferably at least equal tothe MIC₉₀ level (minimum inhibitory concentration, i.e., theconcentration that inhibits the growth of 90% of the target pathogen) ofthe macrolide for the target pathogen. Such an amount may beadministered to the animal recipient in two or more separate doses,although preferably is administered in a single dose. To the extent themacrolide(s) is administered with another active ingredient(s), the term“therapeutically effective amount” refers to the total amounts ofmacrolide and other active ingredient(s) that are together sufficient toprevent, reduce the risk of, delay the onset of, ameliorate, suppress,or eradicate a target pathogen(s) infection.

Factors affecting the preferred dosage regimen include the type (e.g.,species and breed), age, weight, sex, diet, activity, and condition ofthe animal recipient; the severity of the pathological condition; theapparatus used to administer the composition, as well as the type ofadministration used; pharmacological considerations, such as theactivity, efficacy, pharmacokinetic, and toxicology profiles of theparticular composition administered; the existence of an additionalactive ingredient(s) in the composition; and whether the composition isbeing administered as part of a drug and/or vaccine combination. Thus,the dosage actually employed can vary for specific animal patients, and,therefore, can deviate from the typical dosages set forth above.Determining such dosage adjustments is generally within the skill ofthose in the art using conventional means.

In general, the macrolide(s) may be administered once to an animal,although it is contemplated that it may instead be administered multipletimes.

For cattle, the total amount of administered macrolide(s) is typicallyfrom about 0.1 to about 40 mg per kg body weight, and more typicallyfrom about 1 to about 10 mg per kg body weight. For example, in someembodiments, the amount administered to cattle is about 4 mg per kg bodyweight. Although the macrolide(s) may be given to cattle at any age, insome embodiments, the macrolide(s) is administered to cattle that arefrom about 1 months to about 1.5 years old, or from about 6 months toabout 1 year old. In some embodiments, the macrolide(s) is administeredto weaned calves entering the feedlots (often at about 6 months old). Instill other embodiments, the cattle are calves at from about 2 to about12 weeks old, and the macrolide(s) is administered for prophylaxis at adosage of from about 1 to about 10 mg per kg of the body weight; or fortreating an existing infection at a dosage of from about 2 to about 20mg per kg of the body weight.

For swine, the total amount of administered macrolide(s) is typicallyfrom about 0.1 to about 50 mg per kg body weight, and more typicallyfrom about 1 to about 10 mg per kg body weight. For example, in someembodiments, the amount administered to swine is about 4 mg per kg bodyweight. In other embodiments, the amount administered to swine is about5 mg per kg body weight. Although the macrolide(s) may be given to swineat any age, in some embodiments, the macrolide(s) is administered togrower-finisher pigs.

The method of administration can be varied depending on animals, but inthe case of large mammals such as cattle, swine, and horses, it ispreferably administered orally or parenterally. “Parenteraladministration” includes, e.g., subcutaneous injections, intravenousinjections, intramuscular injections, intrasternal injections,submucosal injections, and infusion. In some embodiments, e.g., theanimal recipient is a bovine animal, and the macrolide composition isadministered subcutaneously, such as in the neck. In other embodiments,e.g., the animal recipient is a swine animal, and the macrolidecomposition is administered intramuscularly.

The macrolide(s) may be used to form a pharmaceutical composition (or“medicament”). It is contemplated that such a composition may entirelycomprise one or more macrolides. Normally, however, the compositioncomprises other ingredients as well.

Other ingredients in the composition may comprise, e.g., other activeingredients. Alternatively (or in addition), such other ingredients maycomprise one or more pharmaceutically acceptable carriers, vehicles,and/or adjuvants (collectively referred to as “excipients”). Theselection of such excipients will depend on various factors, such as themode of administration; the apparatus used to administer thecomposition; pharmacological considerations, such as the activity,efficacy, pharmacokinetic, and toxicology profiles of the particularcomposition; the existence of an additional active ingredient(s) in thecomposition; and whether the composition is being administered as partof a drug and/or vaccine combination.

Solid macrolide compositions may comprise, e.g., saccharides such aslactose, glucose, and sucrose; starches, such as corn starch and potatostarch; cellulose derivatives, such as carboxymethylcellulose sodium,ethylcellulose, and cellulose acetate; etc.

Liquid macrolide compositions may comprise for example, water, isotonicphysiological saline, Ringer's solution, ethyl alcohol, and/or phosphatebuffer solution may be present. Such compositions also may compriseoils, such as peanut oil, cotton seed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil and/or polyhydric alcohols such asglycerol, propylene glycol, sorbitol, mannitol, polyethylene glycol, andpoly(ethylene glycol-2-propylene glycol-2-polyethylene glycol). It also,e.g., may be desirable in some instances for the composition to compriseone or more preservatives. The presence of a preservative may, e.g.,provide a benefit for compositions or solvents that may be stored overlengthy periods of time, e.g., days, weeks, months, or years. Whenselecting a suitable preservative, factors to consider include, e.g.,its antimicrobial activity; the pH range at which it has the desiredantimicrobial activity; the minimum concentration at which it has thedesired antimicrobial activity; its aqueous solubility and otherphysical characteristics (e.g., potential to cause foaming); itssuitability for parenteral use; its possible interactions with theactive ingredient(s) (e.g., its effect on the solubility of an activeingredient); its possible interactions with the non-active ingredients(e.g., its effect on the stability of the solvent); and any governmentregulations that may be applicable where the composition or solvent isbeing manufactured, sold, or used. Contemplated preservatives include,e.g., parabens, propylene glycol, benzalkonium chloride, phenylethanol,chlorocresol, metacresol, ethanol, phenoxyethanol, and benzyl alcohol.

Further discussion regarding pharmaceutically acceptable excipients thatmay be suitable for the macrolide composition may be found in, e.g.,“Gennaro, Remington: The Science and Practice of Pharmacy” (20thEdition, 2000) (incorporated by reference herein). To illustrate, othersuitable excipients may include, e.g., coloring agents; flavoringagents; and thickening agents, such as povidone carboxymethylcellulose,and/or hydroxypropyl methylcellulose.

Normally, the macrolide(s) occupies at least about 0.5% by weight of thepharmaceutical composition. For example, in some embodiments for swineuse, suitable macrolide concentrations for parenteral administration maybe, e.g., from about 5 to about 500 mg/ml, from about 10 to about 100mg/ml, or from about 20 to about 60 mg/ml (e.g., about 40 mg/ml).Exemplifying further, in some embodiments for bovine use, suitablemacrolide concentrations for parenteral administration may be, e.g.,from about 5 mg/ml to about 2.0 g/ml, from about 10 mg/ml to about 1.0g/ml, 50 to about 500 mg/ml, or from about 100 to about 300 mg/ml (e.g.,180 mg/ml).

It should be recognized that the macrolide concentration can be varieddepending on the dosage form. Where, e.g., the macrolide(s) isadministered parenterally, the macrolide concentration preferably issufficient to provide the desired therapeutically effective amount ofthe macrolide(s) in a volume that is acceptable for parenteraladministration. The maximum acceptable volume may vary, depending on,e.g., the apparatus being used for the administration, type ofparenteral administration, size of the recipient animal, and subjectivedesires of the user.

In some embodiments, the pharmaceutical composition comprises a liquidcomposition formed by a process comprising dissolving the macrolide(s)in the excipient(s). In other embodiments, the composition comprises asuspension formed by a process comprising suspending the macrolide(s) inthe excipient(s).

Further discussion relating to the use of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide and derivatives thereofto treat livestock and poultry disease may be found in, e.g., U.S. Pat.No. 6,514,946.

This disclosure is also directed to kits that are, e.g., suitable foruse in performing the methods of treatment described above. In someembodiments, the kit comprises a therapeutically effective amount of atleast one of the above-described crystalline forms of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (e.g., a therapeuticallyeffective amount of the Form I polymorph), and instructions forcombining the crystalline form with at least one excipient, such as,e.g., instructions for dissolving or suspending the crystalline form ina liquid excipient. The kit may further (or alternatively) compriseadditional components, such as, e.g., one or more apparatuses (e.g., asyringe) for administering a composition comprising (or derived from)the crystalline form(s) of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, one or more additionalpharmaceutical or biological materials, one or more excipients, and/orone or more diagnostic tools.

EXAMPLES

The following examples are merely illustrative of embodiments of theinvention, and not limiting to the remainder of this disclosure in anyway.

Example 1 Preparation of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolidefrom tylosin A

Part A. Reductive amination. Preparation of23-O-Mycinosyl-20-Piperidinyl-5-O-Mycaminosyl-Tylonolide Compound (2).

Toluene (19.2 kg), tylosin A (1) (3.68 kg; ≧80% tylosin A; ≧95% tylosinA, B, C, & D), piperidine (0.40 kg), and formic acid (0.55 kg) werecharged to a reactor. The mixture was heated to 70-80° C., while beingstirred. Stirring was then continued at that temperature for 1-2 morehours. The formation of the 20-piperidinyl-tylosin compound (2) wasmonitored by HPLC. After reaction completion (≦2% tylosin A (1)), theproduct mixture was cooled to ambient temperature.

Part B. Acid hydrolysis of mycarosyloxy substituent. Preparation of23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound (3).

HBr (48% HBr diluted to 24%) was added to the product mixture of Part Awhile stirring and maintaining the mixture at less than 40° C.Afterward, the phases in the product mixture were separated using a20-minute phase separation period. The product mixture was at 20-25° C.during this phase separation. HPLC of the lower phase was used toconfirm reaction completion (≦2% 20-piperidinyl-tylosin compound (2)).

Part C. Acid hydrolysis of mycinosyloxy substituent. Preparation of23-Hydroxyl-20-Piperidinyl-5-O-Mycaminosyl-Tylonolide (4).

Twenty-four percent HBr (18.4 L) was added at ambient temperature to theaqueous phase obtained from Part B, followed by heating to 54±3° C.within about 1 hour while stirring. Stirring was continued at thistemperature for 2-4 more hours, while the reaction was monitored usingHPLC. After completion of the reaction (≦2%23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound (3)),the mixture was cooled to ambient temperature using a −10° C. coolingjacket. After cooling, the mixture was extracted with dichloromethanethree times (9.8 kg each time). The aqueous product was cooled to 4-8°C., and then 6 N NaOH (33.6 kg) was slowly added to adjust the pH to≧10. The resulting mixture was extracted with dichloromethane threetimes (with 32.6 kg, 29.3 kg, and 24.5 kg) at ambient temperature. Thecombined organic phases were charged to a separate reactor. Sodiumsulfate (2.9 kg; Na₂SO₄) was added and filtered off. Dichloromethane(4.9 kg) was then added and removed via distillation. The resultingcrude product was dissolved and re-crystallized twice in tert-butylmethyl ether (6.1 kg each time) at ambient temperature. Afterward, theproduct was isolated on a Nutsch filter, washed twice with tert-butylmethyl ether (1.0 kg each time), and dried in a tray dryer under vacuumovernight at 40° C. The final product was analyzed using HPLC.

Part D. Iodination. Preparation of activated compound (5).

Triphenylphosphine (0.9 kg) and pyridine (0.3 kg; free of water) weredissolved into dichloromethane (11.7 kg) at ambient temperature. Iodine(0.8 kg) was then added. The resulting mixture was then stirred untilall the iodine dissolved. The mixture was then cooled to 13° C. Thecooled mixture was added to the product from Part C in dichloromethane(11.7 kg) while stirring at 15±3° C. The reaction was monitored by HPLC,and was determined to be completed in 2-2.5 hours (≦2%23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound).

Part E. Amination. Preparation of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (6).

Potassium carbonate (1.8 kg), acetonitrile (16.7 kg), and piperidine(1.1 kg) were added to the product of Part D. The resulting mixture wasthen heated to 78° C. while distilling off dichloromethane. Aftersolvent exchange to acetonitrile, the mixture was stirred at 2-2.5 hoursat reflux, and then cooled to ambient temperature. Afterward, theresidual potassium carbonate was filtered off, the filter cake waswashed with acetonitrile (2.8 kg), and the solvent was distilled offunder vacuum at a 50° C. jacket temperature. The resulting residue wasdissolved in ethyl acetate (15.8 kg), and mixed with 0.5 N HCl (35.6kg). The phases were separated at ambient temperature, and the loweraqueous phase was extracted three times with ethyl acetate (15.8 kg wereused each time). The resulting aqueous phase was set to a pH of 11 byaddition of 6 N NaOH (6.4 kg) and extracted three times withdichloromethane (18.7 kg each time) at ambient temperature. The combinedlower organic phases were recharged to the reactor with sodium sulfate(5.3 kg). The mixture was then filtered to form a cake, which, in turn,was washed with dichloromethane (4.9 kg) and dried under vacuum at ajacket temperature of 50° C. to form a macrolide product. This product,in turn, was mixed with acetonitrile (21.7 L) and re-crystallized. Theresulting crystals were isolated on a Nutsch filter, washed twice withcold acetonitrile (3.5 L each time), and dried under vacuum at 40° C.overnight to form macrolide (5) product. The composition of the productwas confirmed using HPLC.

Example 2 Alternative Amination. Preparation of20,23-Dipiperidinyl-5-O-Mycaminosyl-Tylonolide (2)

Potassium carbonate (0.94 kg), xylene (5 L), and piperidine (0.55 kg)are added to 1.0 kg of activated compound (1) made in accordance withthe procedure in Part D. The resulting mixture is then heated to 95-105°C. for 15 hours. Work-up includes dissolving the K₂CO₃ in water;removing excess piperidine; extracting into diluted HCl; extracting intotert-butyl methyl ether at a pH of 11; conducting a solvent switch toethanol; and precipitating, isolating, and drying of the crude product.The product is then re-crystallized from methyl acetate or ethylacetate. The composition of the product is confirmed using HPLC.

Example 3 Preparing the Form I polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

Part A. Reductive amination. Preparation of23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound (2).

Tylosin phosphate (1) and dichloromethane (1.3 L per kg tylosinphosphate) were charged to a reactor. The resulting mixture was stirredto produce a clear solution. Next, piperidine (1.2 eq, based on thetylosin phosphate), formic acid (4.5 eq, based on the tylosinphosphate), and toluene (6.7 L per kg tylosin phosphate) weresequentially charged to the reactor. The resulting mixture was heated to76° C. while being stirred. Stirring was then continued at thattemperature for 2.5 hours. Additional piperidine (0.1 eq, based on thetylosin phosphate) was then charged, and the resulting mixture wasstirred at 76° C. for an additional hour. The product mixture was cooledto 50° C.

Part B. Acid hydrolysis of mycarosyloxy substituent. Preparation of23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide (4).

Aqueous HBr (23.3 eq, based on the tylosin phosphate used in Part A) wasadded to the product mixture of Part A at 50° C. The resulting mixturewas stirred at 56° C. for 5 hours. HPLC was used to monitor thereaction.

Once the desired conversion was obtained, the product mixture wascooled. The aqueous phase was extracted twice with dichloromethane at25-30° C. The aqueous phase was then cooled to 0° C., and the pH wasadjusted to 10-10.5 with NaOH at ≦5° C. Afterward, the aqueous phase wasextracted twice with dichloromethane at 20° C. The resulting combinedorganic phases were extracted twice with aqueous NaHCO₃. Thedichloromethane was then removed from the combined organic phases viadistillation, and replaced with isopropyl alcohol. Afterward, heptane at45° C. was added to initiate precipitation. The mixture was then stirredat 0° C. Afterward, the crystalline product was isolated by filtration.The isolated crystals were washed with heptane and isopropyl alcohol,dried, and analyzed using HPLC.

The above procedure made 0.23 kg of product per kilogram of tylosinphosphate used in Part A. This product may contain isopropyl alcohol. Toremove the isopropyl alcohol, the product may be dissolved in tolueneand dichloromethane, followed by distillation.

Part C. Iodination. Preparation of activated compound (5).

Triphenylphosphine (0.41 kg per kg of product of Part B) was dissolvedin dichloromethane (12 L per kg of triphenylphosphine, ≦100 ppm H₂O) at25° C. Pyridine (0.3 kg per kg triphenylphosphine) was then added. Next,iodine (0.9 kg per kg of triphenylphosphine) was added in 5 portions at25° C. The resulting mixture was stirred for 40 minutes at 25° C., andthen cooled to −6° C. The mixture was then added to the product fromPart B over 50 minutes while stirring at −6° C. Afterward, stirring wascontinued for 7 hours while maintaining the mixture at −5° C. Thereaction was monitored by HPLC (if sufficient conversion is not reached,the mixture may be stirred at −5° C. for an additional amount of time,e.g., 1.5 hours).

When the desired conversion was reached, the product mixture was washedwith aqueous Na₂SO₃ solution at −5° C. Dichloromethane was then removedfrom the organic phase by distillation, and replaced withtetrahydrofuran.

Part D. Amination. Preparation of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (6).

Piperidine (0.55 kg per kg of product from Part B) was added to theproduct from Part C, followed by potassium carbonate (0.94 kg per kg ofproduct from Part B). The resulting mixture was heated to 55° C., andthen maintained at that temperature for 3 hours while stirring.Afterward, the mixture was heated to 72° C. over 1 hour, and thenstirred at that temperature for 6 hours. The composition of the productwas analyzed using HPLC.

Once the desired conversion was obtained, the product mixture was cooledto 20° C., and toluene was added. The resulting mixture was washed twicewith water, and the organic phase was extracted twice with aqueous HCl,resulting in an aqueous phase having a pH of ≦3. This mixture was cooledto 0-5° C.

Part E. Preparation of ethyl acetate S1 crystalline solvate of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. The acidic aqueousproduct solution prepared in accordance with Part D was combined withethyl acetate (6.7 L per kg of product from Part B) at 3° C. The pH ofthe resulting emulsion was adjusted to 10.5-11.0 at 3° C. with causticsoda. The phases were separated at 3° C. The organic phase was washedonce with water. After phase separation, the organic phase wasconcentrated by distillation, resulting in an ethyl acetate solution.Upon seeding, crystallization began. The resulting product was filteredoff to obtain a filter cake of the ethyl acetate crystalline solvate.The filter cake was washed with heptane at 0° C. This yieldedapproximately 0.78 kg of crude wet crystalline solvate per kg of productfrom Part B used.

Part F. Preparing the Form I polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide. A washed crystallinesolvate wet cake formed in accordance with Part E was combined withheptane (6.1 L per kg wet cake). The resulting suspension was heated to72° C. and seeded. Afterward, the suspension was stirred at 72° C., andthen at 20° C. The suspension was then filtered, and the resultingsolids were washed with heptane and dried. This yielded approximately0.53 kg of Form I crystals per kg of product from Part B used (or 0.68kg of Form I crystals per kg of crude wet crystalline solvate productfrom Part E used).

Example 4 Preparing the Form II polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

Part A. Preparation of activated compound.23-Hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide (50 g) wasprepared in accordance with the process described in Example 1, PartsA-C, except the acid used in the acid hydrolysis reactions (i.e., PartsB and C) was HCl instead of HBr. The23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide at 13° C. wascharged to a stirred reactor containing dichloromethane (250 ml at 13°C.). The resulting mixture was stirred for about 5 minutes at 13° C. Inparallel, dichloromethane (250 ml at ambient temperature) was charged toa separate reactor, and stirring was initiated. Triphenylphosphine (24.6g at ambient temperature) was then charged to the reactor, followed bypyridine (7.8 ml at ambient temperature) and then iodine (22.83 g atambient temperature). Afterward, the mixture was stirred for 2 minutesat ambient temperature, and then combined with the dichloromethanemixture containing 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolideat 13° C. using dropping funnel The resulting mixture was stirred for130 minutes at 13° C. to form an activated product

Part B. Preparing the Form II polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

Potassium carbonate (51.81 g), then acetonitrile (600 ml), and finallypiperidine (37.1 ml) were added to the activated product of Part A at13° C. The resulting mixture was then heated to 78° C. over 90 minutes,and then stirred at that temperature (reflux) for 130 additionalminutes. The mixture was then cooled to 15-25° C. over 60 minutes, andstirring was ceased. Afterward, the residual potassium carbonate wasfiltered off, the filter cake was washed with acetonitrile (100 ml), andthe solvent was distilled off under vacuum at 50° C. over 60 minutes.The resulting residue was dissolved in ethyl acetate (500 ml), and mixedwith 0.5 N HCl (1000 ml). After stirring for 5 minutes, stirring wasceased, and the phases were separated. The lower aqueous phase wasextracted three times with ethyl acetate (500 ml were used each time).Stirring of the resulting aqueous phase was initiated, and thetemperature was reduced to 5-8° C. The pH was then adjusted to a pH of11 by addition of 6 N NaOH (150 ml). The pH-adjusted mixture was thenextracted three times with dichloromethane (400 ml each time) at ambienttemperature. The combined lower organic phases were recharged to thereactor with sodium sulfate (150 g) at ambient temperature. Theresulting mixture was stirred for 15 minutes, and then filtered to forma cake, which, in turn, was washed with dichloromethane (100 ml). Thesolvent was removed by distillation, and the resulting product was driedunder vacuum at 50° C. for 60 minutes. This yielded 57.5 g of crudemacrolide product.

The crude product was crystallized from acetonitrile (90 ml) at 50° C.To avoid oil formation, seeding crystals of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide were added at ambienttemperature (the seeding crystals were obtained earlier by dissolving 3g of crude 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in 12 mlacetonitrile, and collecting via filtration the crystals that formedafter 24 hours at ambient temperature). The product precipitated as anoff-white solid over 5 h at ambient temperature and overnight (15 h) at5° C. The solid was separated by filtration, and washed twice with coldacetonitrile (2×25 ml). The remaining solid was dried under reducedpressure (8 mbar) at 40° C. overnight, resulting in 18.2 g of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (content: 90% (w/w) asdetermined by HPLC). The product (15 g) was further purified byre-crystallization in acetonitrile. This resulted in 10.7 g of product(HPLC purity at 254 nm: 100%; content: 94% (w/w) as determined by HPLC).

Example 5 Re-crystallization of the Form II polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in acetonitrile

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(80 mg) prepared in accordance with Example 4 was dissolved inacetonitrile (2 ml). The resulting solution was filtered, and theacetonitrile was allowed to evaporate at ambient temperature to formcrystals. The FT-Raman spectrum of the product crystals wasapproximately identical to the spectrum of the product crystals inExample 4.

Example 6 Preparing the ethyl acetate S1 crystalline solvate of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(312 mg) prepared in accordance with Example 4 was dissolved in ethylacetate (0.5 ml). A few minutes after complete dissolution, new crystalsformed and, after a few additional minutes, filled the solution.Additional ethyl acetate (1 ml) was added, and the crystals werefiltered off and dried at ambient temperature and atmospheric pressure.

Example 7 Preparing the Form III polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form III polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolidewas prepared by drying, under vacuum at ambient temperature for 20hours, the ethyl acetate S1 crystalline solvate of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (50 mg) prepared inaccordance with Example 6.

Example 8 Preparing the ethyl acetate S1 crystalline solvate of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(146.1 mg) prepared in accordance with Example 4 was dissolved in ethylacetate (0.5 ml) with stirring. After crystallization began, heptane (5ml) was added while stirring was continued. The resulting solid wasfiltered off after 3 days. All these steps were conducted at ambienttemperature. The resulting crystals were in the form of very fineneedles. The FT-Raman spectrum of the crystals coincided with theFT-Raman spectrum of the crystals from Example 6.

Example 9 Preparing the ethyl acetate S1 crystalline solvate of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(99.6 mg) prepared in accordance with Example 4 was dissolved in ethylacetate (2 ml). The resulting solution was filtered, and the solvent wasallowed to evaporate. After evaporation of almost all the solvent, anamorphous reside remained. Ethyl acetate was added again, and allowed toevaporate. A few seed crystals prepared in Example 6 were added atdifferent stages of the evaporation. This yielded crystals in the formof needles. The FT-Raman spectrum of these crystals coincided to theFT-Raman of the crystals from Example 6.

Example 10 Preparing the Form III polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form III polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolidewas prepared by drying, under vacuum at from about 40 to about 70° C.for 3 days, the ethyl acetate S1 crystalline solvate of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide prepared in accordancewith Example 9. The FT-Raman spectrum for these crystals coincided tothe FT-Raman spectrum of the crystals from Example 7.

Example 11 Preparing the Form III polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(150.5 mg) prepared in accordance with Example 4 and acetonitrile (1 ml)were combined and subjected to temperature cycling between 20 and 40° C.with time intervals of 1 hour for each heating/cooling step andtemperature hold. This cycling was stopped after 5 days. The resultingcrystals (in the form of fine needles) were filtered off and allowed todry at ambient temperature. The PXRD spectrum for these crystalscoincided to the PXRD spectrum of the crystals in Example 7.

Example 12 Preparing the Form I polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(170.5 mg) prepared in accordance with Example 4 was stirred at ambienttemperature for 4 days with a solvent (1 ml) consisting of heptane andtert-butyl methyl ether (“tBME”) at a heptane/tBME ratio of 95:5(vol/vol). Afterward, the resulting crystals were filtered off, washedwith additional heptane/tBME (95:5 vol/vol) solvent, and vacuum dried.

Example 13 Preparing the Form I polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(147.4 mg) prepared in accordance with Example 4 was dissolved in tBME(0.5 ml) with stirring to form a clear solution. Heptane was then added,leading to slight precipitation. The crystals were then isolated after 3days. All these steps were conducted at ambient temperature. TheFT-Raman spectrum of the resulting crystals coincided to the FT-Ramanspectrum of the crystals in Example 12.

Example 14 Preparing the Form I polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(164.5 mg) prepared in accordance with Example 4 was stirred withheptane (1 ml) at ambient temperature for 4 days. The resulting solidwas filtered off, washed with heptane, and vacuum dried. The washed anddried product (90 mg) and Form III polymorph crystals of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (98 mg) were suspended inheptane and stirred. The temperature was maintained at 25° C. for 10days, except for an accidental brief temperature increase to 60° C.during the fifth night. The FT-Raman spectrum of the resulting crystalscoincided with the FT-Raman spectrum of the crystals of Example 12.

Example 15 Preparing the Form I polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

Form III polymorph crystals of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (171.8 mg) were suspendedin a solvent (1 ml) of heptane and tBME at a heptane/tBME ratio of 95:5(vol/vol). The resulting solution was stirred at for 9 days. The solidwas filtered off and washed with heptane (1 ml). All these steps wereconducted at ambient temperature. The FT-Raman spectrum of the resultingcrystals coincided with the FT-Raman spectrum of the crystals of Example12.

Example 16 Preparing the Form I polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

Form II polymorph crystals of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (173.4 mg) were suspendedin a solvent (1 ml) of heptane and tBME at a heptane/tBME ratio of 95:5(vol/vol). The resulting solution was stirred for 9 days. The solid wasfiltered off and washed with heptane (1 ml). All these steps wereconducted at ambient temperature. FT-Raman spectra of the crystals at 5days and at the end of the 9 days coincided with the FT-Raman spectrumof the crystals of Example 12.

Example 17 Preparing the ethanol S1 crystalline solvate of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(150 mg) prepared in accordance with Example 4 was dissolved in ethanol(1 ml). After filtering, the ethanol was allowed to evaporate at ambienttemperature. A solid formed, which was once again dissolved in ethanol(1 ml). After filtering, the ethanol was allowed to evaporate at ambienttemperature. The PXRD and FT-Raman spectra for the resulting crystalscoincided with the corresponding spectra for the crystal product ofExample 6.

Example 18 Preparing the diethyl ketone S1 crystalline solvate of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(206.6 mg) prepared in accordance with Example 4 was dissolved indiethyl ketone (0.5 ml), and then allowed to sit overnight. The nextmorning, the crystals were obtained using filtration. The PXRD spectrumfor the resulting crystals coincided with the PXRD spectrum for thecrystal product of Example 6.

Example 19 Preparing the tBME S2 crystalline solvate of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(304 mg) prepared in accordance with Example 4 was dissolved intert-butyl methyl ether (0.5 ml). Overnight, a large crystal formed atthe bottom of the vessel. Upon scratching, the entire solution volumefilled with crystals within 15 minutes. Additional tert-butyl methylether (1 ml) was added. The crystals were then filtered off and dried atambient temperature.

Although this procedure was successfully repeated for forming S2 solvatecrystals, additional batches of S2 solvate crystals were formed bydissolving an additional amount of the Form II polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in tert-butyl methylether, seeding S2 solvate crystals from the first batch, and removingthe tert-butyl methyl ether. In one experiment, the S2 crystallinesolvate was prepared by dissolving the Form II polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (245.7 mg) in tert-butylmethyl ether (0.5 ml), and slowly evaporating a portion of the solventat ambient temperature. After no crystals formed, additional tert-butylmethyl ether was added, followed by seeding S2 solvate crystals from thefirst batch. The solvent was then allowed to evaporate completely. TheFT-Raman spectrum for these crystals was approximately identical to theFT-Raman spectrum for the crystals from the first batch. In furthertesting, the crystals were vacuum-dried at ambient temperature for 20hours, and then dried again under vacuum for 24 hours at about 70° C.The FT-Raman spectra of the crystals after each drying step coincided tothe FT-Raman spectrum from the first batch.

Example 20 Preparing the tetrahydrofuran S3 crystalline solvate of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(150 mg) prepared in accordance with Example 4 was dissolved intetrahydrofuran (1.0 ml). The resulting mixture was filtered, and thenthe solvent was allowed to evaporate at ambient temperature.Crystallization occurred after a relatively large proportion of thesolvent evaporated.

Example 21 Preparing the methyl acetate S4 crystalline solvate of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(204.0 mg) prepared in accordance with Example 4 was dissolved in methylacetate (0.5 ml). Re-crystallization initiated during dissolution. After15 minutes, the whole volume was filled with needles. The solid wasfiltered off. The final crystals were prismatic in shape.

Example 22 Preparing the ethyl formate S4 crystalline solvate of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

The Form II polymorph of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide(208.3 mg) prepared in accordance with Example 4 was dissolved in ethylformate (0.5 ml). The flask was left open for a few minutes, whereuponthe material slowly crystallized to form large needles. The solid wasfiltered off. The final crystals were prismatic in shape. The PXRDspectrum for the resulting crystals coincided with the PXRD spectrum forthe crystal product of Example 21.

Example 23 Preparing the Form IV polymorph of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide

Solvent-wet ethyl acetate 51 crystalline solvate of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (3.4 g, corresponding to2.0 g dry product) was mixed with 27.7 g heptane (which corresponds to aratio of 14 g solvent to 1 g product). The mixture was distilled at73-95° C. to remove 8.4 g of solvent (ethyl acetate and heptanecombined), which also resulted in a product dissolution. The solutionwas cooled to 45° C. within 2 hours, which lead to precipitation of somesticky solid at 45° C. The solution was heated to 60° C., and seedingcrystals were added (these seeding crystals were prepared earlier bymixing crude 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide (0.9 g) withheptane (4.5 g), stirring the mixture at 80° C. for 8 hours, stirringthe mixture at 23° C. for 21 hours, and filtering off the resultingcrystals). The solution was cooled to 45° C., whereupon some solidformed. The mixture was heated to 80° C., and then maintained at thattemperature while being stirred for 8 hours. Afterward, the mixture wascooled to 22° C., causing product to form at the wall of the reactionflask. This product was separated.

* * *

The words “comprise,” “comprises,” and “comprising” in this disclosure(including the claims) are to be interpreted inclusively rather thanexclusively.

The term “pharmaceutically acceptable” is used adjectivally in thisdisclosure to mean that the modified noun is appropriate for use in apharmaceutical product. When it is used, e.g., to describe an excipientor a salt, it characterizes the excipient or salt as having a benefit(s)that outweighs any deleterious effect(s) that the excipient or salt mayhave to the intended recipient animal.

Unless otherwise characterized by this disclosure, the term “ambienttemperature” means a temperature of from about 20 to about 25° C.

The term “amorphous” as applied to20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in this disclosure refersto a solid-state wherein the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide molecules are present ina disordered arrangement, and do not form a distinguishable crystallattice or unit cell. When subjected to powder X-ray diffraction,amorphous 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide does notproduce any characteristic crystalline peaks.

The term “crystalline form” as applied to20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide in this disclosure refersto a solid-state form wherein the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide molecules are arranged toform a distinguishable crystal lattice that: (i) comprisesdistinguishable unit cells, and (ii) yields diffraction peaks whensubjected to powder X-ray radiation.

The term “crystallization” can refer to crystallization and/orre-crystallization, depending on the applicable circumstances relatingto preparation of the 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolidestarting material.

The term “direct crystallization” refers to crystallization of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide directly from a suitablesolvent without formation and desolvation of an intermediate solvatedcrystalline solid-state form of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

The term “particle size” refers to particle size, as measured byconventional particle size measuring techniques well known in the art,such as laser light scattering, sedimentation field flow fractionation,photon correlation spectroscopy, or disk centrifugation. A non-limitingexample of a technique that can be used to measure particle size is aliquid dispersion technique employing a Sympatec Particle Size Analyzer.

The term “HPLC” means high pressure liquid chromatography.

Unless otherwise characterized by this disclosure, the term “purity”means the chemical purity of20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide according to conventionalHPLC assay.

The term “phase purity” as used in this disclosure means the solid-statepurity of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide with regard toa particular crystalline or amorphous form of the20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide as determined by X-raypowder diffraction analytical methods described in this disclosure. Theterm “phase-pure” refers to purity with respect to other solid-stateforms of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide, and does notnecessarily imply a high degree of chemical purity with respect to othercompounds. The term “substantially phase-pure” refers to at least about90% purity (e.g., at least about 95% purity) with respect to othersolid-state forms of 20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide.

All references cited in this disclosure are incorporated by referenceherein.

What is claimed is:
 1. A crystalline macrolide or a salt thereofproduced by a process, wherein the macrolide corresponds in structure toFormula (I):

wherein the process comprises: reacting tylosin A or a salt thereof, apiperidinyl compound of Formula (II), and formic acid in a presence of anon-polar solvent to form a 20-piperidinyl-tylosin compound, reactingthe 20-piperidinyl-tylosin compound with an acid to faun a23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound,reacting the 23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound with an acid to form a23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound,activating the 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound with an activating agent to form an activated compound,reacting the activated compound with a piperidinyl compound of Formula(VII) to provide a crude product; and crystallizing the crude product toproduce a crystalline macrolide; wherein the piperidinyl compound ofFormula (II) corresponds in structure to:

the 20-piperidinyl-tylosin compound corresponds in structure to Formula(III):

the 23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compoundcorresponds in structure to Formula (IV):

the 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compoundcorresponds in structure to Formula (V):

the activated compound corresponds in structure to Formula (VI):

the piperidinyl compound of Formula (VII) corresponds in structure to:

L is a leaving group; and as to R¹, R², and R³: R¹ and R³ are eachmethyl, and R² is hydrogen, R¹ and R³ are each hydrogen, and R² ismethyl, or R¹, R², and R³ are each hydrogen; and as to R⁴, R⁵, and R⁶:R⁴ and R⁶ are each methyl, and R⁵ is hydrogen, R⁴ and R⁶ are eachhydrogen, and R⁵ is methyl, or R⁴, R⁵, and R⁶ are each hydrogen, whereinthe crystalline macrolide or salt thereof has a melting point of fromabout 192 to about 195° C. and at least one of the followingcharacteristics: an FT-Raman spectrum comprising an absorption band atone or more frequencies selected from the group consistint of about2935, about 1633, about 1596, about 1712, about 1683, and about 781cm⁻¹, wherein the FT-Raman spectrum is substantially as shown in FIG. 2;a powder X-ray diffraction spectrum comprising at least one peakselected from the group consisting of 5.0 (±0.2) and 5.6 (±0.2) degrees2Θ, wherein the powder X-ray diffraction spectrum is substantially asshown in FIG. 1; or an attenuated total reflection infrared spectrumcomprising an absorption band at one or more frequencies selected fromthe group consisting of about 2932, about 1711, about 1682, about 1635,about 1599, about 1442, about 1404, about 1182, about 1079, about 1053,about 1008, about 985, about 842, and about 783 cm⁻¹, wherein theattenuated total reflection infrared spectrum is substantially as shownin FIG.
 6. 2. The crystalline macrolide or salt thereof of claim 1having: an FT-Raman spectrum comprising an absorption band at one ormore frequencies selected from the group consisting of about 2935, about1633, about 1596, about 1712, about 1683, and about 781 cm⁻¹; a powderX-ray diffraction spectrum comprising at least one peak selected fromthe group consisting of 5.0 (±0.2) and 5.6 (±0.2) degrees 2Θ; and anattenuated total reflection infrared spectrum comprising an absorptionband at one or more frequencies selected from the group consisting ofabout 2932, about 1711, about 1682, about 1635, about 1599, about 1442,about 1404, about 1182, about 1079, about 1053, about 1008, about 985,about 842, and about 783 cm⁻¹.
 3. The crystalline macrolide or saltthereof of claim 1 having at least one of the following characteristics:an FT-Raman spectrum comprising an absorption band at one or morefrequencies selected from the group consisting of about 2935 and about1633 cm⁻¹; a powder X-ray diffraction spectrum comprising a peak at 5.0(±0.2) degrees 2Θ; or an attenuated total reflection infrared spectrumcomprising an absorption band at one or more frequencies selected fromthe group consisting of about 1711, about 1682, about 1635, about 1599,about 1404, about 1182, and about 783 cm⁻¹.
 4. The crystalline macrolideor salt thereof of claim 3 having at least one of the followingcharacteristics: an FT-Raman spectrum comprising an absorption band atabout 1633 cm⁻¹; a powder X-ray diffraction spectrum comprising a peakat 5.0(±0.2) degrees 2Θ; or an attenuated total reflection infraredspectrum comprising an absorption band at one or more frequenciesselected from the group consisting of about 1711, about 1682, about1635, about 1599, about 1404, about 1182, and about 783 cm⁻¹.
 5. Thecrystalline macrolide or salt thereof of claim 4 having an attenuatedtotal reflection infrared spectrum comprising an absorption band at oneor more frequencies selected from the group consisting of about 1711 andabout 1682 cm⁻¹.
 6. The crystalline macrolide or salt thereof of claim 4having an attenuated total reflection infrared spectrum comprising anabsorption band at one or more frequencies selected from the groupconsisting of about 1635, about 1404, and about 1182 cm⁻¹.
 7. Acrystalline macrolide or salt thereof, wherein the macrolide correspondsin structure to Formula (I):

wherein the process comprises: reacting tylosin A or a salt thereof, apiperidinyl compound of Formula (II), and formic acid in a presence of anon-polar solvent to form a 20-piperidinyl-tylosin compound, reactingthe 20-piperidinyl-tylosin compound with an acid to form a23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound,reacting the 23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound with an acid to form a23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound,activating the 23-hydroxl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound with an activating agent to form an activated compound,reacting the activated compound with a piperidinyl compound of Formula(VII) to provide a crude product; and crystallizing the crude product toproduce a crystalline macrolide; wherein the piperidinyl compound ofFormula (II) corresponds in structure to:

the 20-piperidinyl-tylosin compound corresponds in structure to Formula(III):

the 23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compoundcorresponds in structure to Formula (IV):

the 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compoundcorresponds in structure to Formula (V):

the activated compound corresponds in structure to Formula (VI):

the piperidinyl compound of Formula (VII) corresponds in structure to:

L is a leaving group; and as to R¹, R², and R³: R¹ and R³ are eachmethyl, and R² is hydrogen, R¹ and R³ are each hydrogen, and R² ismethyl, or R¹, R², and R³ are each hydrogen; and as to R⁴, R⁵, and R⁶:R⁴ and R⁶ are each methyl, and R⁵ is hydrogen, R⁴ and R⁶ are eachhydrogen, and R⁵ is methyl, or R⁴, R⁵, and R⁶ are each hydrogen, whereinthe crystalline macrolide or salt thereof is a20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide form having at least oneof the following characteristics: an FT-Raman spectrum comprising anabsorption band at one or more frequencies selected from the groupconsisting of about 2929, about 1625, about 1595, about 1685, and about783 cm⁻¹, wherein the FT-Raman spectrum is substantially as shown inFIG. 9; a powder X-ray diffraction spectrum comprising a peak at 6.5(±0.2) degrees 2Θ, wherein the powder X-ray diffraction spectrum issubstantially as shown in FIG. 8; or an attenuated total reflectioninfrared spectrum comprising an absorption band at one or morefrequencies selected from the group consisting of about 2935, about1736, about 1668, about 1587, about 1451, about 1165, about 1080, about1057, about 1042, about 1005, about 981, about 838, and about 755 cm⁻¹,wherein the attenuated total reflection infrared spectrum issubstantially as shown in FIG.
 13. 8. A crystalline macrolide or saltthereof, wherein the macrolide corresponds in structure to Formula (I):

wherein the process comprises: reacting tylosin A or a salt thereof, apiperidinyl compound of Formula (II), and formic acid in a presence of anon-polar solvent to form a 20-piperidinyl-tylosin compound, reactingthe 20-piperidinyl-tylosin compound with an acid to form a23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound,reacting the 23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound with an acid to form a23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound,activating the 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound with an activating agent to form an activated compound,reacting the activated compound with a piperidinyl compound of Formula(VII) to provide a crude product; and crystallizing the crude product toproduce a crystalline macrolide; wherein the piperidinyl compound ofFormula (II) corresponds in structure to:

the 20-piperidinyl-tylosin compound corresponds in structure to Formula(III):

the 23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compoundcorresponds in structure to Formula (IV):

the 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compoundcorresponds in structure to Formula (V):

the activated compound corresponds in structure to Formula (VI):

the piperidinyl compound of Formula (VII) corresponds in structure to:

L is a leaving group; and as to R¹, R², and R³: R¹ and R³ are eachmethyl, and R² is hydrogen, R¹ and R³ are each hydrogen, and R² ismethyl, or R¹, R², and R³ are each hydrogen; and as to R⁴, R⁵, and R⁶:R⁴ and R⁶ are each methyl, and R⁵ is hydrogen, R⁴ and R⁶ are eachhydrogen, and R⁵ is methyl, or R⁴, R⁵, and R⁶ are each hydrogen, whereinthe crystalline macrolide or salt thereof is a20,23-dipiperidinyl-5-O-mycaminosyl-tylonolide form having at least oneof the following characteristics: an FT-Raman spectrum comprising anabsorption band at one or more frequencies selected from the groupconsisting of about 2943, about 2917, about 1627, about 1590, about1733, about 1669, about 1193, about 1094, and about 981 cm⁻¹, whereinthe FT-Raman spectrum is substantially as shown in FIG. 16; a powderX-ray diffraction spectrum comprising at least one peak selected fromthe group consisting of 5.6 (±0.2) and 6.1 (±0.2) degrees 2Θ, whereinthe powder X-ray diffraction spectrum is substantially as shown in FIG.15; or an attenuated total reflection infrared spectrum comprising anabsorption band at one or more frequencies selected from the groupconsisting of about 2931, about 1732, about 1667, about 1590, about1453, about 1165, about 1081, about 1057, about 1046, about 1005, about981, about 834, and about 756 cm⁻¹, wherein the attenuated totalreflection infrared spectrum is substantially as shown in FIG.
 20. 9. Acrystalline macrolide or salt thereof, wherein the macrolide correspondsin structure to Formula (I):

wherein the process comprises: reacting tylosin A or a salt thereof, apiperidinyl compound of Formula (II), and formic acid in a presence of anon-solar solvent to form a 20- piperidinyl-tylosin compound, reactingthe 20-piperidinyl-tylosin compound with an acid to form a23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound,reacting the 23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound with an acid to form a23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compound,activating the 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolidecompound with an activating agent to form an activated compound,reacting the activated compound with a piperidinyl compound of Formula(VII) to provide a crude product; and crystallizing the crude product toproduce a crystalline macrolide; wherein the piperidinyl compound ofFormula (II) corresponds in structure to:

the 20-piperidinyl-tylosin compound corresponds in structure to Formula(III):

the 23-O-mycinosyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compoundcorresponds in structure to Formula (IV):

the 23-hydroxyl-20-piperidinyl-5-O-mycaminosyl-tylonolide compoundcorresponds in structure to Formula (V):

the activated compound corresponds in structure to Formula (VI):

the piperidinyl compound of Formula (VII) corresponds in structure to:

L is a leaving group; and as to R¹, R², and R³: R¹ and R³ are eachmethyl, and R² is hydrogen, R¹ and R³ are each hydrogen, and R² ismethyl, or R¹, R², and R³ are each hydrogen; and as to R⁴, R⁵, and R⁶:R⁴ and R⁶ are each methyl, and R⁵ is hydrogen, R⁴ and R⁶ are eachhydrogen, and R⁵ is methyl, or R⁴, R⁵, and R⁶ are each hydrogen, whereinthe crystalline macrolide or salt thereof is a20,23-dipiperidinyl-5-O-mycaminosy-tylonolide form having the followingcharacteristics: an attenuated total reflection infrared spectrumcomprising an absorption band at one or more frequencies selected fromthe group consisting of about 3559, about 2933, about 1743, about 1668,about 1584, about 1448, about 1165, about 1075, about 1060, about 1045,about 1010, about 985, about 839, and about 757 cm⁻¹, wherein theattenuated total reflection infrared spectrum is substantially as shownin FIG. 24; and a melting point of from about 149 to about 155° C.
 10. Amethod for treating a disease in an animal, the disease selected fromthe group consisting of pasteurellosis, swine respiratory disease, andbovine respiratory disease; the method comprising: administering thecrystalline macrolide or acceptable salt of claim 1 to the animal. 11.The method according to claim 10, wherein the disease is selected fromthe group consisting of swine respiratory disease associated with atleast one of Actinobacillus pleuropneumoniae, Pasteurella multocida, andBordetella bronchiseptica, and bovine respiratory disease associatedwith at least one of Mannheimia haemolytica, Pasteurella multocida, andHistophilus somni.
 12. The method according to claim 11, wherein themethod further comprises: forming the crystalline macrolide oracceptable salt into a suspension, and administering the suspension tothe animal.